Anti-glyco-muc1 antibodies and their uses

ABSTRACT

The present disclosure relates to anti-glyco-MUC1 antibodies and antigen binding fragments thereof that specifically bind to a cancer-specific glycosylation variant of MUC1 and related fusion proteins and antibody-drug conjugates, as well as nucleic acids encoding such biomolecules. The present disclosure further relates to use of the antibodies, antigen-binding fragments, fusion proteins, antibody-drug conjugates and nucleic acids for cancer therapy.

1. CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 16/168,259,filed Oct. 23, 2018, which claims the benefit of U.S. provisionalapplication Nos. 62/575,666, filed Oct. 23, 2017, and 62/576,297, filedOct. 24, 2017, the contents of which are incorporated herein in theirentireties by reference thereto.

2. SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 23, 2018 isnamed GOT-001D1_Sequence_Listing.txt and is 33,267 bytes in size.

3. BACKGROUND

The human mucin MUC1 is a polymorphic transmembrane glycoproteinexpressed on the apical surfaces of simple and glandular epithelia(Taylor-Papadimitriou et al., 1999). MUC1 is highly overexpressed andaberrantly O-glycosylated in adenocarcinomas. The extracellular domainof the mucin contains variable number of tandem repeats (TRs) (25-125)of 20 amino acid residues with five potential sites for O-glycosylation.O-Glycans are incompletely processed in cancer cells resulting in theexpression of the pancarcinoma carbohydrate antigens Tn(GalNAcα1-O-Ser/Thr) (Springer, 1984). Simple mucin-type O-glycans, Tn,are widely expressed in adenocarcinomas (including breast and ovariancancers) and show limited distribution in normal adult tissues(Springer, 1984). The expression of these O-glycans in cancer correlateswith poor prognosis and natural antibodies to these carbohydrate haptensincreases in cancer patients (Miles et al., 1995; Soares et al., 1996;Werther et al., 1996). There is a need in the art for therapeuticmodalities that utilize glyco-MUC1 epitopes that are overexpressed incancer cells.

4. SUMMARY

The disclosure captures the tumor specificity of glycopeptide variantsby providing therapeutic and diagnostic agents based on antibodies andantigen binding fragments that are selective for cancer-specificepitopes of glyco-MUC1.

The present disclosure provides anti-glyco-MUC1 antibodies and antigenbinding fragments thereof that bind to a cancer-specific glycosylationvariant of MUC1. The present disclosure further provides fusion proteinsand antibody-drug conjugates comprising anti-glyco-MUC1 antibodies andantigen binding fragments, and nucleic acids encoding theanti-glyco-MUC1 antibodies, antigen binding fragments and fusionproteins.

The present disclosure further provides methods of using theanti-glyco-MUC1 antibodies, antigen-binding fragments, fusion proteins,antibody-drug conjugates and nucleic acids for cancer therapy.

In certain aspects, the disclosure provides bispecific and othermultispecific anti-glyco-MUC1 antibodies and antigen binding fragmentsthat bind to a cancer-specific glycosylation variant of MUC1 and to asecond epitope. The second epitope can either be on MUC1 itself, onanother protein co-expressed on cancer cells with MUC1, or on anotherprotein presented on a different cell, such as an activated T cell.Further, also disclosed are nucleic acids encoding such antibodies,including nucleic acids comprising codon-optimized coding regions andnucleic acids comprising coding regions that are not codon-optimized forexpression in a particular host cell.

The anti-glyco-MUC1 antibodies and binding fragments can be in the formof fusion proteins containing a fusion partner. The fusion partner canbe useful to provide a second function, such as a signaling function ofthe signaling domain of a T cell signaling protein, a peptide modulatorof T cell activation or an enzymatic component of a labeling system.Exemplary T cell signaling proteins include 4-1BB, CO3C, and fusionpeptides, e.g., CD28-CD3-zeta and 4-IBB-CD3-zeta. 4-1BB, or CD137, is aco-stimulatory receptor of T cells; CD3-zeta is a signal-transductioncomponent of the T-cell antigen receptor. The moiety providing a secondfunction can be a modulator of T cell activation, such as IL-15,IL-15Ra, or an IL-15/IL-15Ra fusion, or it can encode a label or anenzymatic component of a labeling system useful in monitoring the extentand/or location of binding in vivo or in vitro. Constructs encodingthese prophylactically and therapeutically active biomolecules placed inthe context of T cells, such as autologous T cells, provide a powerfulplatform for recruiting adoptively transferred T cells to prevent ortreat a variety of cancers in some embodiments of the disclosure.

In certain aspects, an anti-glyco-MUC1 antibody or antigen-bindingfragment of the disclosure comprises heavy and/or light chain variablesequences (or encoded by the nucleotide sequences) set forth in Table 1.For clarity, when the term “anti-glyco-MUC1 antibody” is used in thisdocument, it is intended to include monospecific and multi-specific(including bispecific) anti-glyco-MUC1 antibodies, antigen-bindingfragments of the monospecific and multi-specific antibodies, and fusionproteins and conjugates containing the antibodies and theirantigen-binding fragments, unless the context dictates otherwise.Likewise, when the term when the term “anti-glyco-MUC1 antibody orantigen-binding fragment” is used, it is also intended to includemonospecific and multi-specific (including bispecific) anti-glyco-MUC1antibodies and their antigen-binding fragments, together with fusionproteins and conjugates containing such antibodies and antigen-bindingfragments, unless the context dictates otherwise.

In other aspects, an anti-glyco-MUC1 antibody or antigen-bindingfragment of the disclosure comprises heavy and/or light chain CDRsequences (or encoded by the nucleotide sequences) set forth in Tables1-3. The CDR sequences set forth in Table 1 include CDR sequencesdefined according to the IMGT (Lefranc et al., 2003, Dev ComparatImmunol 27:55-77, Kabat (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md.), and Chothia (Al-Lazikani et al.,1997, J. Mol. Biol 273:927-948) schemes for defining CDR boundaries. TheCDR sequences set forth in Table 2 are the combined regions of overlapfor the CDR sequences shown in Table 1, with the IMGT, Kabat and Chothiasequences shown in underlined bold text. The CDR sequences set forth inTable 3 are the common regions of overlap for the CDR sequences shown inTable 1. The framework sequences for such anti-glyco-MUC1 antibody andantigen-binding fragment can be the native murine framework sequences inTable 1 or can be non-native (e.g., humanized or human) frameworksequences.

TABLE 1 SEQ Description Sequence ID NO: VH amino acidMGWSGIFLFFLSVTTGVHSQVQLQQSDAELVKPGASVK  1 sequence (incl.ISCKASGYTFTDHAIHWVKQRPEQGLEWIGYFSPGNDD signalIHYNEKFEGKATLTADKSSSTAYMQLNSLTSEDSAVYF sequence) CKRSYDKDFDCWGQGTTLTVSSVL amino acid MVLILLLLWVSGTCGDIVMSQSPSSLGVSVGEKVTMSC  2 sequence (incl.KSSQSLLYSTNQKNYQSLLYSTNQKNYLAWYQQKPGQS signalPKLLIYWVSNRKSGVPDRFTGSGSGTDFTLTISSVKAE sequence)DLAVYYCQQYYRYPLTFGAGTKLELK VH amino acidQVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVK  3 sequenceQRPEQGLEWIGYFSPGNDDIHYNEKFEGKATLTADKSS (predictedSTAYMQLNSLTSEDSAVYFCKRSYDKDFDCWGQGTTLT mature) VSS VL amino acidDIVMSQSPSSLGVSVGEKVTMSCKSSQSLLYSTNQKNY  4 sequenceQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWVSNRKSGV (predictedPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYRYPL mature) TFGAGTKLELK CDR-H1 aminoGYTFTDHA  5 acid sequence (IMGT definition) CDR-H2 amino FSPGNDDI  6acid sequence (IMGT definition) CDR-H3 amino KRSYDKDFDC  7 acid sequence(IMGT definition) CDR-L1 amino QSLLYSTNQKNY  8 acid sequence (IMGTdefinition) CDR-L2 amino WVS  9 acid sequence (IMGT definition)CDR-L3 amino QQYYRYPLT 10 acid sequence (IMGT definition) VH nucleotideATGGGATGGAGCGGGATCTTTCTCTTCTTCCTGTCAGT 11 sequence (incl.AACTACAGGTGTCCACTCCCAGGTTCAGCTGCAGCAGT signalCTGACGCGGAGTTGGTGAAACCTGGGGCTTCAGTGAAG sequence)ATATCCTGCAAGGCTTCTGGCTACACTTTCACTGACCATGCTATTCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATTTTTCTCCCGGAAATGATGACATTCACTACAATGAGAAGTTCGAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAACAGCCTGACATCTGAAGATTCTGCAGTGTATTTCTGTAAAAGATCTTACGACAAGGACTTTGACTGCTGGGG CCAAGGCACCACTCTCACAGTCTCCTCAVL nucleotide ATGGTTCTTATCTTACTGCTGCTATGGGTATCTGGTAC 12 sequence (incl.CTGTGGGGACATTGTGATGTCACAGTCTCCATCCTCCC signalTAGGTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGC sequence)AAGTCCAGTCAGAGCCTTTTATACAGTACCAATCAAAAGAACTACCTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAGTTGCTGATTTACTGGGTATCTAATAGGAAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGGTATCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCT GAAA VH nucleotideCAGGTTCAGCTGCAGCAGTCTGACGCGGAGTTGGTGAA 13 sequence (excl.ACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTG signalGCTACACTTTCACTGACCATGCTATTCACTGGGTGAAG sequence)CAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATTTTTCTCCCGGAAATGATGACATTCACTACAATGAGAAGTTCGAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAACAGCCTGACATCTGAAGATTCTGCAGTGTATTTCTGTAAAAGATCTTACGACAAGGACTTTGACTGCTGGGGCCAAGGCACCACTCTCACA GTCTCCTCA VL nucleotideGACATTGTGATGTCACAGTCTCCATCCTCCCTAGGTGT 14 sequence (excl.GTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCA signalGTCAGAGCCTTTTATACAGTACCAATCAAAAGAACTAC sequence)CTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAGTTGCTGATTTACTGGGTATCTAATAGGAAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGGTATCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA FR-H1 QVQLQQSDAELVKPGASVKISCKAS 15FR-H2 IHWVKQRPEQGLEWIGY 16 FR-H3 HYNEKFEGKATLTADKSSSTAYMQLNSLTSEDSAVYFC17 FR-H4 WGQGTTLTVSS 18 FR-L1 DIVMSQSPSSLGVSVGEKVTMSCKSS 19 FR-L2LAWYQQKPGQSPKLLIY 20 FR-L3 NRKSGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYC 21 FR-L4FGAGTKLELK 22 CDR-H1 amino DHAIH 23 acid sequence (Kabat definition)CDR-H2 amino YFSPGNDDIHYNEKFEG 24 acid sequence (Kabat definition)CDR-H3 amino SYDKDFDC 25 acid sequence (Kabat definition) CDR-L1 aminoKSSQSLLYSTNQKNYLA 26 acid sequence (Kabat definition) CDR-L2 aminoWVSNRKS 27 acid sequence (Kabat definition) CDR-L3 amino QQYYRYPLT 10acid sequence (Kabat definition) CDR-H1 amino GYTFTDH 28 acid sequence(Chothia definition) CDR-H2 amino SPGNDD 29 acid sequence (Chothiadefinition) CDR-H3 amino SYDKDFDC 25 acid sequence (Chothia definition)CDR-L1 amino SQSLLYSTNQKNY 30 acid sequence (Chothia definition)CDR-L2 amino WVS  9 acid sequence (Chothia definition) CDR-L3 aminoYYRYPLT 31 acid sequence (Chothia definition)

TABLE 2 SEQ Description Sequence ID NO: CDR-H1 amino GYTFTDHA IH (IMGT)32 acid sequence GYTFT DHAIH  (Kabat) (combined GYTFTDH AIH (Chothia)overlap) CDR-H2 amino Y FSPGNDDI HYNEKFEG (IMGT) 24 acid sequenceYFSPGNDDIHYNEKFEG  (Kabat) (combined YF SPGNDD IHYNEKFEG (Chothia)overlap) CDR-H3 amino KRSYDKDFDC  (IMGT)  7 acid sequence KR SYDKDFDC (Kabat) (combined KR SYDKDFDC  (Chothia) overlap) CDR-L1 amino KSSQSLLYSTNQKNY LA (IMGT) 26 acid sequence KSSQSLLYSTNQKNYLA  (Kabat)(combined KS SQSLLYSTNQKNY LA (Chothia) overlap) CDR-L2 amino WVSNRKS (IMGT) 27 acid sequence WVSNRKS  (Kabat) (combined WVSNRKS (Chothia) overlap) CDR-L3 amino QQYYRYPLT  (IMGT) 10 acid sequenceQQYYRYPLT  (Kabat) (combined QQ YYRYPLT  (Chothia) overlap)

TABLE 3 SEQ Description Sequence ID NO: CDR-H1 amino DH 33 acid sequence(common sequence) CDR-H2 amino SPGNDD 29 acid sequence (common sequence)CDR-H3 amino SYDKDFDC 25 acid sequence (common sequence) CDR-L1 aminoQSLLYSTNQKNY  8 acid sequence (common sequence) CDR-L2 amino WVS  9acid sequence (common sequence) CDR-L3 amino YYRYPLT 31 acid sequence(common sequence)

In certain aspects, the disclosure provides an anti-glyco-MUC1 antibodyor antigen-binding fragment of the disclosure comprises CDRs comprisingthe amino acid sequences of any of the CDR combinations set forth innumbered embodiments 3 to 17. Thus, in certain embodiments, ananti-glyco-MUC1 antibody or antigen-binding fragment of the disclosurecomprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:33, aCDR-H2 comprising the amino acid sequence of SEQ ID NO:29, a CDR-H3comprising the amino acid sequence of SEQ ID NO:25, a CDR-L1 comprisingthe amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the aminoacid sequence of SEQ ID NO:9, and a CDR-L3 comprising the amino acidsequence of SEQ ID NO:31. In some embodiments, CDR-H1 comprises theamino acid sequence of SEQ ID NO: 5, 23, 28, or 32. In some embodiments,CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6 or 24. In someembodiments, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7.In some embodiments, CDR-L1 comprises the amino acid sequence of SEQ IDNO:30 or 26. In some embodiments, CDR-L2 comprises the amino acidsequence of SEQ ID NO:27. In some embodiments, CDR-L3 comprises theamino acid sequence of SEQ ID NO:10. In other aspects, ananti-glyco-MUC1 antibody or antigen-binding fragment of the disclosurecomprises heavy chain CDRs of SEQ ID NOS: 5-7 and light chain CDRs ofSEQ ID NOS: 8-10. In other aspects, an anti-glyco-MUC1 antibody orantigen-binding fragment of the disclosure comprises heavy chain CDRs ofSEQ ID NOS: 23-25 and light chain CDRs of SEQ ID NOS: 26, 27, and 10. Inother aspects, an anti-glyco-MUC1 antibody or antigen-binding fragmentof the disclosure comprises heavy chain CDRs of SEQ ID NOS: 28, 29, and25 and light chain CDRs of SEQ ID NOS: 30, 9, and 31. In other aspects,an anti-glyco-MUC1 antibody or antigen-binding fragment of thedisclosure comprises heavy chain CDRs of SEQ ID NOS: 32, 24, and 7 andlight chain CDRs of SEQ ID NOS: 26, 27, and 10. In other aspects, ananti-glyco-MUC1 antibody or antigen-binding fragment of the disclosurecomprises heavy chain CDRs of SEQ ID NOS: 33, 29, and 25 and light chainCDRs of SEQ ID NOS: 8, 9, and 31. The antibody or antigen-bindingfragment can be murine, chimeric, humanized or human.

In further aspects, an anti-glyco-MUC1 antibody or antigen bindingfragment of the disclosure competes with an antibody or antigen bindingfragment comprising heavy and light chain variable regions of SEQ IDNOS: 3 and 4, respectively. In yet other aspects, the disclosureprovides an anti-MUC1 antibody or antigen binding fragment having heavyand light chain variable regions having at least 95%, 98%, 99%, or 99.5%sequence identity of SEQ ID NOS: 3 and 4, respectively.

In yet other aspects, an anti-glyco-MUC1 antibody or antigen-bindingfragment of the disclosure is a single-chain variable fragment (scFv).An exemplary scFv comprises the heavy chain variable fragment N-terminalto the light chain variable fragment. In some embodiments, the scFvheavy chain variable fragment and light chain variable fragment arecovalently bound to a linker sequence of 4-15 amino acids. The scFv canbe in the form of a bi-specific T-cell engager or within a chimericantigen receptor (CAR).

The anti-glyco-MUC1 antibodies and antigen-binding fragments can be inthe form of a multimer of a single-chain variable fragment, a bispecificsingle-chain variable fragment and a multimer of a bispecificsingle-chain variable fragment. In some embodiments, the multimer of asingle chain variable fragment is selected a divalent single-chainvariable fragment, a tribody or a tetrabody. In some of theseembodiments, the multimer of a bispecific single-chain variable fragmentis a bispecific T-cell engager.

Other aspects of the disclosure are drawn to nucleic acids encoding theanti-glyco-MUC1 antibodies and antibody-binding fragments of thedisclosure. In some embodiments, the portion of the nucleic acid nucleicacid encoding an anti-glyco-MUC1 antibody or antigen-binding fragment iscodon-optimized for expression in a human cell. In certain aspects, thedisclosure provides an anti-glyco-MUC1 antibody or antigen bindingfragment having heavy and light chain variable regions encoded by aheavy chain nucleotide sequence having at least 95%, 98%, 99%, or 99.5%sequence identity to SEQ ID NO:11 or SEQ ID NO:13 and a light chainnucleotide sequence having at least 95%, 98%, 99%, or 99.5% sequenceidentity to SEQ ID NO:12 or SEQ ID NO:14. Vectors (e.g., a viral vectorsuch as a lentiviral vector) and host cells comprising the nucleic acidsare also within the scope of the disclosure. The heavy and light chainscoding sequences can be present on a single vector or on separatevectors.

Yet another aspect of the disclosure is a pharmaceutical compositioncomprising an anti-glyco-MUC1 antibody, antigen-binding fragment,nucleic acid (or pair of nucleic acids), vector (or pair or vectors) orhost cell according to the disclosure, and a physiologically suitablebuffer, adjuvant or diluent.

Still another aspect of the disclosure is a method of making a chimericantigen receptor comprising incubating a cell comprising a nucleic acidor a vector according to the disclosure, under conditions suitable forexpression of the coding region and collecting the chimeric antigenreceptor.

Another aspect of the disclosure is a method of detecting cancercomprising contacting a cell or tissue sample with an anti-glyco-MUC1antibody or antigen-binding fragment of the disclosure and detectingwhether the antibody is bound to the cell or tissue sample.

Yet another aspect of the disclosure is a method of treating cancercomprising administering a prophylactically or therapeutically effectiveamount of an anti-glyco-MUC1 antibody, antigen-binding fragment, nucleicacid, vector, host cell or pharmaceutical composition according to thedisclosure to a subject in need thereof.

5. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Results of ELISA assay showing specificity of binding of GO2 toglyco-MUC1 relative to MUC1.

FIG. 2: Binding of GO2 to colon cancer tissue. Immunohistochemistrylabeling of invasive colon carcinoma tissue and adjacent healthy tissueusing mAbs GO2. mAb GO2 shows distinct binding to colon cancer tissuewith high reactivity with both intracellular and surface structures oncancer cells. In contrast no reactivity is seen to surface structures onhealthy colon cells.

FIG. 3: Binding of GO2 to pancreatic cancer tissue. Immunohistochemistrylabeling of pancreatic cancer tissue using mAbs GO2. mAb GO2 showdistinct binding to pancreatic cancer cells. In contrast no or limitedreactivity is seen to surrounding healthy tissue.

FIG. 4: Binding of GO2 to breast cancer tissue. Immunohistochemistrylabeling of breast cancer tissue using mAbs GO2. mAb GO2 showed distinctbinding to invasive breast cancer cells.

FIG. 5: Results of an antibody dependent cellular cytotoxicity assaywith antibody GO2 and a secondary antibody conjugated to the antitubulinagent monomethyl auristatin F (MMAF).

FIG. 6: Results of an ELISA assay quantifying circulating tumor cellsusing GO2. X-axis shows number of cells and Y-axis shows OD450 values.

FIGS. 7A-E: Representative images of MUC1 positive TMA tumor cores. FIG.7A: breast cancer; FIG. 7B: non-small cell lung cancer; FIG. 7C: ovariancancer; FIG. 7D: colorectal cancer; FIG. 7E: prostate cancer.

FIG. 8: Schematic of an exemplary anti-glyco-MUC1 and anti-CD3 T-cellbispecific antibody (TCB).

FIGS. 9A-B: Jurkat-NFAT activation assay with undigested patient-derivedtumor samples (malignant neoplasm of bronchus and lung: middle lobe,bronchus or lung, squamous cell carcinoma) and different TCBs at 50 nM(FIG. 9A) or 5 nM (FIG. 9B).

FIG. 10: Jurkat-NFAT activation assay with undigested patient-derivedtumor samples (malignant neoplasm of bronchus and lung: lower lobe,bronchus or lung, non-keratinizing squamous cell carcinoma) anddifferent TCBs at 50 nM.

FIG. 11: Jurkat-NFAT activation assay with undigested patient-derivedtumor samples (malignant neoplasm of bronchus and lung: upper lobe,bronchus or lung, adenocarcinoma with acinar type) and different TCBs at50 nM.

FIGS. 12A-12B: Binding of GO2 TCB to MUC1 expressed on MCF7 cs (FIG.12A) and T3M4 pzfv (FIG. 12B) cells measured by flow cytometry.

FIGS. 13A-X: Induction of tumor cell killing and T cell activationmeasured by upregulation of CD25 and CD69 on CD4 T cells and CD8 T cellsas well as release of IL6, IL8, IL10, IFNγ, TNFα and Granzyme B with GO2TCB on T3M4 pzfv in the presence of PBMCs from two healthy donors (donor1 FIG. 13A-13L; donor 2 FIG. 13M-13X). Same legend for each of FIGS.13A-13X.

FIGS. 14A-14F: Induction of tumor cell killing (FIGS. 14A-14B) and Tcell activation measured by upregulation of CD25 and CD69 on CD8 T cellsand CD4 T cells (FIGS. 14C-14F, respectively) with GO2 TCB on MCF7 cs inthe presence of PBMCs. Same legend for each of FIGS. 14A-14F.

FIG. 15A-B: Binding of GO2 TCB and HMFG1 TCB to MCF10A (humannon-tumorigenic mammary epithelial cell line) (FIG. 15A) and HBEpiC(human bronchial epithelial cells) (FIG. 15B).

FIG. 16A-C: Induction of tumor cell killing (FIG. 16A) and T cellactivation measured by upregulation of CD25 on CD4 T cells (FIG. 16B)and CD8 T cells (FIG. 16C) with GO2 TCB and HMFG1 TCB on MCF10A cells inthe presence of PBMCs.

FIG. 17: Illustration of GO2 and GO2 TCB flowing through a flow cellhaving coupled glycopeptides.

FIG. 18A-B: Sensorgrams showing binding of GO2 (FIG. 18A) and GO2 TCB(FIG. 18B) to human and cynomolgous glycopeptides.

FIG. 19A-D: Binding (avidity) of GO2 antibody (FIG. 19A-19B) and GO2 TCB(FIG. 19C-19D) to human and cynomolgus glycopeptides, and estimate ofthe “apparent” KD.

6. DETAILED DESCRIPTION 6.1 Antibodies

The inventor has developed novel antibodies that are directed to aglycoform of MUC1 present on tumor cells. These are exemplified by theantibody 5F7, referred to herein as “GO2”. GO2 was identified in ascreen for antibodies that bind to a glycosylated 60-mer representing 3copies of one of the tandem repeats present in MUC1,VTSAPDTRPAPGSTAPPAHG (SEQ ID NO:50), glycosylated with purifiedrecombinant human glycosyltransferases polypeptides GalNAc-T2,GalNAc-T4, and GalNAc-T1 so as to mimic the glycosylation pattern ofMUC1 present on tumor cells.

The anti-glyco-MUC1 antibodies of the disclosure, exemplified byantibody GO2, are useful as tools in cancer diagnosis and therapy.

Thus, in certain aspects, the disclosure provides antibodies and antigenbinding fragments that bind to a glycoform of MUC1 present on tumorcells (referred to herein as “glyco-MUC1”), and preferably to the 60-merpeptide (VTSAPDTRPAPGSTAPPAHG)₃ (SEQ ID NO:47) glycosylated withGalNAc-T2, GalNAc-T4, and GalNAc-T1 as described in U.S. Pat. No.6,465,220.

The anti-glyco-MUC1 antibodies of the disclosure may be polyclonal,monoclonal, genetically engineered, and/or otherwise modified in nature,including but not limited to chimeric antibodies, humanized antibodies,human antibodies, primatized antibodies, single chain antibodies,bispecific antibodies, dual-variable domain antibodies, etc. In variousembodiments, the antibodies comprise all or a portion of a constantregion of an antibody. In some embodiments, the constant region is anisotype selected from: IgA (e.g., IgA₁ or IgA₂), IgD, IgE, IgG (e.g.,IgG₁, IgG₂, IgG₃ or IgG₄), and IgM. In specific embodiments, theanti-glyco-MUC1 antibodies of the disclosure comprise an IgG₁ constantregion isotyope.

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. A monoclonal antibodyis derived from a single clone, including any eukaryotic, prokaryotic,or phage clone, by any means available or known in the art. Monoclonalantibodies useful with the present disclosure can be prepared using awide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof. In many uses of the present disclosure, including in vivo useof the anti-glyco-MUC1 antibodies in humans, chimeric, primatized,humanized, or human antibodies can suitably be used.

The term “chimeric” antibody as used herein refers to an antibody havingvariable sequences derived from a non-human immunoglobulin, such as arat or a mouse antibody, and human immunoglobulin constant regions,typically chosen from a human immunoglobulin template. Methods forproducing chimeric antibodies are known in the art. See, e.g., Morrison,1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechniques4:214-221; Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporatedherein by reference in their entireties.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins that contain minimal sequences derived from non-humanimmunoglobulin. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin sequence. The humanizedantibody can also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin consensussequence. Methods of antibody humanization are known in the art. See,e.g., Riechmann et al., 1988, Nature 332:323-7; U.S. Pat. Nos.5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370 to Queen etal.; EP239400; PCT publication WO 91/09967; U.S. Pat. No. 5,225,539;EP592106; EP519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnickaet al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. Natl.Acad. Sci. 91:969-973; and U.S. Pat. No. 5,565,332, all of which arehereby incorporated by reference in their entireties.

“Human antibodies” include antibodies having the amino acid sequence ofa human immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries or from animals transgenic for one or morehuman immunoglobulin and that do not express endogenous immunoglobulins.Human antibodies can be made by a variety of methods known in the artincluding phage display methods using antibody libraries derived fromhuman immunoglobulin sequences. See U.S. Pat. Nos. 4,444,887 and4,716,111; and PCT publications WO 98/46645; WO 98/50433; WO 98/24893;WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741, each of which isincorporated herein by reference in its entirety. Human antibodies canalso be produced using transgenic mice which are incapable of expressingfunctional endogenous immunoglobulins but which can express humanimmunoglobulin genes. See, e.g., PCT publications WO 98/24893; WO92/01047; WO 96/34096; WO 96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;5,916,771; and 5,939,598, which are incorporated by reference herein intheir entireties. Fully human antibodies that recognize a selectedepitope can be generated using a technique referred to as “guidedselection.” In this approach, a selected non-human monoclonal antibody,e.g., a mouse antibody, is used to guide the selection of a completelyhuman antibody recognizing the same epitope (see, Jespers et al., 1988,Biotechnology 12:899-903).

“Primatized antibodies” comprise monkey variable regions and humanconstant regions. Methods for producing primatized antibodies are knownin the art. See, e.g., U.S. Pat. Nos. 5,658,570; 5,681,722; and5,693,780, which are incorporated herein by reference in theirentireties.

Anti-glyco-MUC1 antibodies of the disclosure include both full-length(intact) antibody molecules, as well as antigen-binding fragments thatare capable of binding glyco-MUC1. Examples of antigen-binding fragmentsinclude by way of example and not limitation, Fab, Fab′, F (ab′)₂, Fvfragments, single chain Fv fragments and single domain fragments.

A Fab fragment contains the constant domain of the light chain (CL) andthe first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. F(ab′) fragments are producedby cleavage of the disulfide bond at the hinge cysteines of the F(ab′)₂pepsin digestion product. Additional chemical couplings of antibodyfragments are known to those of ordinary skill in the art. Fab andF(ab′)₁ fragments lack the Fc fragment of intact antibody, clear morerapidly from the circulation of animals, and may have less non-specifictissue binding than an intact antibody (see, e.g., Wahl et al., 1983, J.Nucl. Med. 24:316).

An “Fv” fragment is the minimum fragment of an antibody that contains acomplete target recognition and binding site. This region consists of adimer of one heavy and one light chain variable domain in a tight,non-covalent association (V_(H)-V_(L) dimer). It is in thisconfiguration that the three CDRs of each variable domain interact todefine a target binding site on the surface of the V_(H)-V_(L) dimer.Often, the six CDRs confer target binding specificity to the antibody.However, in some instances even a single variable domain (or half of anFv comprising only three CDRs specific for a target) can have theability to recognize and bind target, although at a lower affinity thanthe entire binding site.

“Single-chain Fv” or “scFv” antigen-binding fragments comprise the V_(H)and V_(L) domains of an antibody, where these domains are present in asingle polypeptide chain. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the scFv to form the desired structure for target binding.

“Single domain antibodies” are composed of single V_(H) or V_(L) domainswhich exhibit sufficient affinity to glyco-MUC1. In a specificembodiment, the single domain antibody is a camelized antibody (See,e.g., Riechmann, 1999, Journal of Immunological Methods 231:25-38)

The anti-glyco-MUC1 antibodies of the disclosure may also be bispecificand other multiple specific antibodies. Bispecific antibodies aremonoclonal, often human or humanized, antibodies that have bindingspecificities for two different epitopes on the same or differentantigen. In the present disclosure, one of the binding specificities canbe directed towards glyco-MUC1, the other can be for any other antigen,e.g., for a cell-surface protein, receptor, receptor subunit,tissue-specific antigen, virally derived protein, virally encodedenvelope protein, bacterially derived protein, or bacterial surfaceprotein, etc. In certain preferred embodiments, the bispecific and othermultispecific anti-glyco-MUC1 antibodies and antigen binding fragmentsthat specifically bind to a second MUC1 epitope, an epitope on anotherprotein co-expressed on cancer cells with MUC1, or an epitope on anotherprotein presented on a different cell, such as an activated T cell.Bispecific antibodies of the disclosure include IgG format bispecificantibodies and single chain-based bispecific antibodies.

IgG format bispecific antibodies of the disclosure can be any of thevarious types of IgG format bispecific antibodies known in the art, suchas quadroma bispecific antibodies, “knobs-in-holes” bispecificantibodies, CrossMab bispecific antibodies, charge paired bispecificantibodies, common light chain bispecific antibodies, one-armsingle-chain Fab-immunoglobulin gamma bispecific antibodies, disulfidestabilized Fv bispecific antibodies, DuetMabs, controlled Fab-armexchange bispecific antibodies, strand-exchange engineered domain bodybispecific antibodies, two-arm leucine zipper heterodimeric monoclonalbispecific antibodies, KA-body bispecific antibodies, dual variabledomain bispecific antibodies, and cross-over dual variable domainbispecific antibodies. See, e.g., Köhler and Milstein, 1975, Nature256:495-497; Milstein and Cuello, 1983, Nature 305:537-40; Ridgway etal., 1996, Protein Eng. 9:617-621; Schaefer et al., 2011, Proc Natl AcadSci USA 108:11187-92; Gunasekaran et al., 2010, J Biol Chem285:19637-46; Fischer et al., 2015 Nature Commun 6:6113; Schanzer etal., 2014, J Biol Chem 289:18693-706; Metz et al., 2012 Protein Eng DesSel 25:571-80; Mazor et al., 2015 MAbs 7:377-89; Labrijn et al., 2013Proc Natl Acad Sci USA 110:5145-50; Davis et al., 2010 Protein Eng DesSel 23:195-202; Wranik et al., 2012, J Biol Chem 287:43331-9; Gu et al.,2015, PLoS One 10(5):e0124135; Steinmetz et al., 2016, MAbs 8(5):867-78;Klein et al., 2016, mAbs, 8(6):1010-1020; Liu et al., 2017, Front.Immunol. 8:38; and Yang et al., 2017, Int. J. Mol. Sci. 18:48, which areincorporated herein by reference in their entireties.

In some embodiments, the bispecific antibodies of the disclosure areCrossMabs. The CrossMab technology is described in detail in WO2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254. WO2013/026833, WO 2016/020309, and Schaefer et al., 2011, Proc Natl AcadSci USA 108:11187-92, which are incorporated herein by reference intheir entireties. Briefly, the CrossMab technology is based on a domaincrossover between heavy and light chains within one Fab-arm of abispecific IgG, which promotes correct chain association. A CrossMabbispecific antibody of the disclosure can be a “CrossMab^(FAB)”antibody, in which the heavy and light chains of the Fab portion of onearm of a bispecific IgG antibody are exchanged. In other embodiments, aCrossMab bispecific antibody of the disclosure can be a“CrossMab^(VH-VL)” antibody, in which the only the variable domains ofthe heavy and light chains of the Fab portion of one arm of a bispecificIgG antibody are exchanged. In yet other embodiments, a CrossMabbispecific antibody of the disclosure can be a “CrossMab^(CH1-CL)”antibody, in which only the constant domains of the heavy and lightchains of the Fab portion of one arm of a bispecific IgG antibody areexchanged. CrossMab^(CH1-CL) antibodies, in contrast to CrossMab^(FAB)and CrossMab^(VH-VL), do not have predicted side products and,therefore, in some embodiments CrossMab^(CH1-CL) bispecific antibodiesare preferred. See, Klein et al., 2016, mAbs, 8(6):1010-1020. Furtherembodiments of CrossMabs of the disclosure are described below inSection 6.2.

In some embodiments, the bispecific antibodies of the disclosure arecontrolled Fab-arm exchange bispecific antibodies. Methods for makingFab-arm exchange bispecific antibodies are described in PCT PublicationNo. WO2011/131746 and Labrijn et al., 2014 Nat Protoc. 9(10):2450-63,incorporated herein by reference in their entireties. Briefly,controlled Fab-arm exchange bispecific antibodies can be made byseparately expressing two parental IgG1s containing single matchingpoint mutations in the CH3 domain, mixing the parental IgG1s under redoxconditions in vitro to enable recombination of half-molecules, andremoving the reductant to allow reoxidation of interchain disulfidebonds, thereby forming the bispecific antibodies.

Bispecific antibodies of the disclosure can comprise an Fc domaincomposed of a first and a second subunit. In one embodiment, the Fcdomain is an IgG Fc domain. In a particular embodiment, the Fc domain isan IgG₁ Fc domain. In another embodiment the Fc domain is an IgG₄ Fcdomain. In a more specific embodiment, the Fc domain is an IgG₄ Fcdomain comprising an amino acid substitution at position S228 (Kabat EUindex numbering), particularly the amino acid substitution S228P. Thisamino acid substitution reduces in vivo Fab arm exchange of IgG₄antibodies (see Stubenrauch et al., 2010, Drug Metabolism andDisposition 38:84-91). In a further particular embodiment, the Fc domainis a human Fc domain. In an even more particular embodiment, the Fcdomain is a human IgG₁ Fc domain. An exemplary sequence of a human IgG₁Fc region is given in SEQ ID NO:42.

In particular embodiments, the Fc domain comprises a modificationpromoting the association of the first and the second subunit of the Fcdomain. The site of most extensive protein-protein interaction betweenthe two subunits of a human IgG Fc domain is in the CH3 domain. Thus, inone embodiment said modification is in the CH3 domain of the Fc domain.

In a specific embodiment said modification promoting the association ofthe first and the second subunit of the Fc domain is a so-called“knob-into-hole” modification, comprising a “knob” modification in oneof the two subunits of the Fc domain and a “whole” modification in theother one of the two subunits of the Fc domain. The knob-into-holetechnology is described e.g. in U.S. Pat. Nos. 5,731,168; 7,695,936;Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, J, 2001, ImmunolMeth 248:7-15. Generally, the method involves introducing a protuberance(“knob”) at the interface of a first polypeptide and a correspondingcavity (“hole”) in the interface of a second polypeptide, such that theprotuberance can be positioned in the cavity so as to promoteheterodimer formation and hinder homodimer formation. Protuberances areconstructed by replacing small amino acid side chains from the interfaceof the first polypeptide with larger side chains (e.g. tyrosine ortryptophan). Compensatory cavities of identical or similar size to theprotuberances are created in the interface of the second polypeptide byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine).

Accordingly, in some embodiments, an amino acid residue in the CH3domain of the first subunit of the Fc domain is replaced with an aminoacid residue having a larger side chain volume, thereby generating aprotuberance within the CH3 domain of the first subunit which ispositionable in a cavity within the CH3 domain of the second subunit,and an amino acid residue in the CH3 domain of the second subunit of theFc domain is replaced with an amino acid residue having a smaller sidechain volume, thereby generating a cavity within the CH3 domain of thesecond subunit within which the protuberance within the CH3 domain ofthe first subunit is positionable. Preferably said amino acid residuehaving a larger side chain volume is selected from the group consistingof arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (VV).Preferably said amino acid residue having a smaller side chain volume isselected from the group consisting of alanine (A), serine (S), threonine(T), and valine (V). The protuberance and cavity can be made by alteringthe nucleic acid encoding the polypeptides, e.g. by site-specificmutagenesis, or by peptide synthesis. An exemplary substitution isY470T.

In a specific such embodiment, in the first subunit of the Fc domain thethreonine residue at position 366 is replaced with a tryptophan residue(T366W), and in the second subunit of the Fc domain the tyrosine residueat position 407 is replaced with a valine residue (Y407V) and optionallythe threonine residue at position 366 is replaced with a serine residue(T366S) and the leucine residue at position 368 is replaced with analanine residue (L368A) (numbering according to Kabat EU index). In afurther embodiment, in the first subunit of the Fc domain additionallythe serine residue at position 354 is replaced with a cysteine residue(S354C) or the glutamic acid residue at position 356 is replaced with acysteine residue (E356C) (particularly the serine residue at position354 is replaced with a cysteine residue), and in the second subunit ofthe Fc domain additionally the tyrosine residue at position 349 isreplaced by a cysteine residue (Y349C) (numbering according to Kabat EUindex). In a particular embodiment, the first subunit of the Fc domaincomprises the amino acid substitutions S354C and T366W, and the secondsubunit of the Fc domain comprises the amino acid substitutions Y349C,T366S, L368A and Y407V (numbering according to Kabat EU index).

In some embodiments, electrostatic steering (e.g., as described inGunasekaran et al., 2010, J Biol Chem 285(25):19637-46) can be used topromote the association of the first and the second subunit of the Fcdomain.

In some embodiments, the Fc domain comprises one or more amino acidsubstitutions that reduces binding to an Fc receptor and/or effectorfunction.

In a particular embodiment the Fc receptor is an Fcγ receptor. In oneembodiment the Fc receptor is a human Fc receptor. In one embodiment theFc receptor is an activating Fc receptor. In a specific embodiment theFc receptor is an activating human Fcγ receptor, more specifically humanFcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In oneembodiment the effector function is one or more selected from the groupof complement dependent cytotoxicity (CDC), antibody-dependentcell-mediated cytotoxicity (ADCC), antibody-dependent cellularphagocytosis (ADCP), and cytokine secretion. In a particular embodiment,the effector function is ADCC.

Typically, the same one or more amino acid substitution is present ineach of the two subunits of the Fc domain. In one embodiment, the one ormore amino acid substitution reduces the binding affinity of the Fcdomain to an Fc receptor. In one embodiment, the one or more amino acidsubstitution reduces the binding affinity of the Fc domain to an Fcreceptor by at least 2-fold, at least 5-fold, or at least 10-fold.

In one embodiment, the Fc domain comprises an amino acid substitution ata position selected from the group of E233, L234, L235, N297, P331 andP329 (numberings according to Kabat EU index). In a more specificembodiment, the Fc domain comprises an amino acid substitution at aposition selected from the group of L234, L235 and P329 (numberingsaccording to Kabat EU index). In some embodiments, the Fc domaincomprises the amino acid substitutions L234A and L235A (numberingsaccording to Kabat EU index). In one such embodiment, the Fc domain isan IgG₁ Fc domain, particularly a human IgG₁ Fc domain. In oneembodiment, the Fc domain comprises an amino acid substitution atposition P329. In a more specific embodiment, the amino acidsubstitution is P329A or P329G, particularly P329G (numberings accordingto Kabat EU index). In one embodiment, the Fc domain comprises an aminoacid substitution at position P329 and a further amino acid substitutionat a position selected from E233, L234, L235, N297 and P331 (numberingsaccording to Kabat EU index). In a more specific embodiment, the furtheramino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D orP331S. In particular embodiments, the Fc domain comprises amino acidsubstitutions at positions P329, L234 and L235 (numberings according toKabat EU index). In more particular embodiments, the Fc domain comprisesthe amino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA”or “LALAPG”). Specifically, in particular embodiments, each subunit ofthe Fc domain comprises the amino acid substitutions L234A, L235A andP329G (Kabat EU index numbering), i.e. in each of the first and thesecond subunit of the Fc domain the leucine residue at position 234 isreplaced with an alanine residue (L234A), the leucine residue atposition 235 is replaced with an alanine residue (L235A) and the prolineresidue at position 329 is replaced by a glycine residue (P329G)(numbering according to Kabat EU index). In one such embodiment, the Fcdomain is an IgG₁ Fc domain, particularly a human IgG₁ Fc domain.

Single chain-based bispecific antibodies of the disclosure can be any ofthe various types of single chain-based bispecific antibodies known inthe art, such as bispecific T-cell engagers (BiTEs), diabodies, tandamdiabodies (tandabs), dual-affinity retargeting molecules (DARTs), andbispecific killer cell engagers. See, e.g., Löffler et al., 2000, Blood95:2098-103; Holliger et al., 1993, Proc Natl Acad Sci USA, 90:6444-8;Kipriyanov et al., 1999, Mol Biol 293:41-56; Johnson et al., 2010, MolBiol 399:436-49; Wiernik et al., 2013, Clin Cancer Res 19:3844-55; Liuet al., 2017, Front. Immunol. 8:38; and Yang et al., 2017, Int. J. Mol.Sci. 18:48, which are incorporated herein by reference in theirentireties.

In some embodiments, the bispecific antibodies of the disclosure arebispecific T-cell engagers (BiTEs). BiTEs are single polypeptide chainmolecules that having two antigen-binding domains, one of which binds toa T-cell antigen and the second of which binds to an antigen present onthe surface of a target (See, PCT Publication WO 05/061547; Baeuerle etal., 2008, Drugs of the Future 33: 137-147; Bargou, et al., 2008,Science 321:974-977, incorporated herein by reference in theirentireties). Thus, the BiTEs of the disclosure have an antigen bindingdomain that binds to a T-cell antigen, and a second antigen bindingdomain that is directed towards glyco-MUC1.

In some embodiments, the bispecific antibodies of the disclosure aredual-affinity retargeting molecules (DARTs). DARTs comprise at least twopolypeptide chains that associate (especially through a covalentinteraction) to form at least two epitope binding sites, which mayrecognize the same or different epitopes. Each of the polypeptide chainsof a DART comprise an immunoglobulin light chain variable region and animmunoglobulin heavy chain variable region, but these regions do notinteract to form an epitope binding site. Rather, the immunoglobulinheavy chain variable region of one (e.g., the first) of the DARTpolypeptide chains interacts with the immunoglobulin light chainvariable region of a different (e.g., the second) DART™ polypeptidechain to form an epitope binding site. Similarly, the immunoglobulinlight chain variable region of one (e.g., the first) of the DARTpolypeptide chains interacts with the immunoglobulin heavy chainvariable region of a different (e.g., the second) DART polypeptide chainto form an epitope binding site. DARTs may be monospecific, bispecific,trispecific, etc., thus being able to simultaneously bind one, two,three or more different epitopes (which may be of the same or ofdifferent antigens). DARTs may additionally be monovalent, bivalent,trivalent, tetravalent, pentavalent, hexavalent, etc., thus being ableto simultaneously bind one, two, three, four, five, six or moremolecules. These two attributes of DARTs (i.e., degree of specificityand valency may be combined, for example to produce bispecificantibodies (i.e., capable of binding two epitopes) that are tetravalent(i.e., capable of binding four sets of epitopes), etc. DART moleculesare disclosed in PCT Publications WO 2006/113665, WO 2008/157379, and WO2010/080538, which are incorporated herein by reference in theirentireties.

In some embodiments of the bispecific antibodies of the disclosure, oneof the binding specificities is directed towards glyco-MUC1, and theother is directed to an antigen expressed on immune effector cells. Theterm “immune effector cell” or “effector cell” as used herein refers toa cell within the natural repertoire of cells in the mammalian immunesystem which can be activated to affect the viability of a target cell.Immune effector cells include cells of the lymphoid lineage such asnatural killer (NK) cells, T cells including cytotoxic T cells, or Bcells, but also cells of the myeloid lineage can be regarded as immuneeffector cells, such as monocytes or macrophages, dendritic cells andneutrophilic granulocytes. Hence, said effector cell is preferably an NKcell, a T cell, a B cell, a monocyte, a macrophage, a dendritic cell ora neutrophilic granulocyte. Recruitment of effector cells to aberrantcells means that immune effector cells are brought in close vicinity tothe aberrant target cells such that the effector cells can directlykill, or indirectly initiate the killing of the aberrant cells that theyare recruited to. In order to avoid non specific interactions it ispreferred that the bispecific antibodies of the disclosure specificallyrecognize antigens on immune effector cells that are at leastover-expressed by these immune effector cells compared to other cells inthe body. Target antigens present on immune effector cells may includeCD3, CD8, CD16, CD25, CD28, CD64, CD89, NKG2D and NKp46. Preferably, theantigen on immune effector cells is CD3 expressed on T cells.

As used herein, “CD3” refers to any native CD3 from any vertebratesource, including mammals such as primates (e.g. humans), non-humanprimates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats),unless otherwise indicated. The term encompasses “full-length,”unprocessed CD3 as well as any form of CD3 that results from processingin the cell. The term also encompasses naturally occurring variants ofCD3, e.g., splice variants or allelic variants. The most preferredantigen on an immune effector cell is the CD3 epsilon chain. Thisantigen has been shown to be very effective in recruiting T cells toaberrant cells. Hence, a bispecific antibody of the disclosurepreferably specifically recognizes CD3 epsilon. The amino acid sequenceof human CD3 epsilon is shown in UniProt (www.uniprot.org) accession no.P07766 (version 144), or NCBI (www.ncbi.nlm.nih.gov/) RefSeqNP_000724.1. The amino acid sequence of cynomolgus [Macaca fascicularis]CD3 epsilon is shown in NCBI GenBank no. BAB71849.1. For humantherapeutic use, bispecific antibodies in which the CD3-binding domainspecifically binds to human CD3 (e.g., the human CD3 epsilon chain) areused. For preclinical testing in non-human animals and cell lines,bispecific antibodies in which the CD3-binding domain specifically bindsto the CD3 in the species utilized for the preclinical testing (e.g.,cynomolgus CD3 for primate testing) can be used.

As used herein, a binding domain that “specifically binds to” or“specifically recognizes” a target antigen from a particular speciesdoes not preclude the binding to or recognition of the antigen fromother species, and thus encompasses antibodies in which one or more ofthe binding domains have inter-species cross-reactivity. For example, aCD3-binding domain that “specifically binds to” or “specificallyrecognizes” human CD3 may also bind to or recognize cyomolgus CD3, andvice versa.

In some embodiments, a bispecific antibody of the disclosure can competewith monoclonal antibody H2C (described in PCT publication no.WO2008/119567) for binding an epitope of CD3. In other embodiments, abispecific antibody of the disclosure can compete with monoclonalantibody V9 (described in Rodrigues et al., 1992, Int J Cancer Suppl7:45-50 and U.S. Pat. No. 6,054,297) for binding an epitope of CD3. Inyet other embodiments, a bispecific antibody of the disclosure cancompete with monoclonal antibody FN18 (described in Nooij et al., 1986,Eur J Immunol 19:981-984) for binding an epitope of CD3. In yet otherembodiments, a bispecific antibody of the disclosure can compete withmonoclonal antibody SP34 (described in Pessano et al., 1985, EMBO J4:337-340) for binding an epitope of CD3.

The anti-glyco-MUC1 antibodies of the disclosure include derivatizedantibodies. For example, but not by way of limitation, derivatizedantibodies are typically modified by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein. Any of numerous chemical modifications can becarried out by known techniques, including, but not limited to, specificchemical cleavage, acetylation, formylation, metabolic synthesis oftunicamycin, etc. Additionally, the derivative can contain one or morenon-natural amino acids, e.g., using ambrx technology (See, e.g.,Wolfson, 2006, Chem. Biol. 13(10):1011-2).

The anti-glyco-MUC1 antibodies or binding fragments may be antibodies orfragments whose sequences have been modified to alter at least oneconstant region-mediated biological effector function. For example, insome embodiments, an anti-glyco-MUC1 antibody may be modified to reduceat least one constant region-mediated biological effector functionrelative to the unmodified antibody, e.g., reduced binding to the Fcreceptor (FcγR). FcγR binding can be reduced by mutating theimmunoglobulin constant region segment of the antibody at particularregions necessary for FcγR interactions (See, e.g., Canfield andMorrison, 1991, J. Exp. Med. 173:1483-1491; and Lund et al., 1991, J.Immunol. 147:2657-2662). Reduction in FcγR binding ability of theantibody can also reduce other effector functions which rely on FcγRinteractions, such as opsonization, phagocytosis and antigen-dependentcellular cytotoxicity (“ADCC”).

The anti-glyco-MUC1 antibody or binding fragments described hereininclude antibodies and/or binding fragments that have been modified toacquire or improve at least one constant region-mediated biologicaleffector function relative to an unmodified antibody, e.g., to enhanceFcγR interactions (See, e.g., US 2006/0134709). For example, ananti-glyco-MUC1 antibody of the disclosure can have a constant regionthat binds FcγRIIA, FcγRIIB and/or FcγRIIIA with greater affinity thanthe corresponding wild type constant region.

Thus, antibodies of the disclosure may have alterations in biologicalactivity that result in increased or decreased opsonization,phagocytosis, or ADCC. Such alterations are known in the art. Forexample, modifications in antibodies that reduce ADCC activity aredescribed in U.S. Pat. No. 5,834,597. An exemplary ADCC lowering variantcorresponds to “mutant 3” (shown in FIG. 4 of U.S. Pat. No. 5,834,597)in which residue 236 is deleted and residues 234, 235 and 237 (using EUnumbering) are substituted with alanines.

In some embodiments, the anti-glyco-MUC1 antibodies of the disclosurehave low levels of, or lack, fucose. Antibodies lacking fucose have beencorrelated with enhanced ADCC activity, especially at low doses ofantibody. See Shields et al., 2002, J. Biol. Chem. 277:26733-26740;Shinkawa et al., 2003, J. Biol. Chem. 278:3466-73. Methods of preparingfucose-less antibodies include growth in rat myeloma YB2/0 cells (ATCCCRL 1662). YB2/0 cells express low levels of FUT8 mRNA, which encodesα-1,6-fucosyltransferase, an enzyme necessary for fucosylation ofpolypeptides.

In yet another aspect, the anti-glyco-MUC1 antibodies or bindingfragments include modifications that increase or decrease their bindingaffinities to the fetal Fc receptor, FcRn, for example, by mutating theimmunoglobulin constant region segment at particular regions involved inFcRn interactions (see, e.g., WO 2005/123780). In particularembodiments, an anti-glyco-MUC1 antibody of the IgG class is mutatedsuch that at least one of amino acid residues 250, 314, and 428 of theheavy chain constant region is substituted alone, or in any combinationsthereof, such as at positions 250 and 428, or at positions 250 and 314,or at positions 314 and 428, or at positions 250, 314, and 428, withpositions 250 and 428 a specific combination. For position 250, thesubstituting amino acid residue can be any amino acid residue other thanthreonine, including, but not limited to, alanine, cysteine, asparticacid, glutamic acid, phenylalanine, glycine, histidine, isoleucine,lysine, leucine, methionine, asparagine, proline, glutamine, arginine,serine, valine, tryptophan, or tyrosine. For position 314, thesubstituting amino acid residue can be any amino acid residue other thanleucine, including, but not limited to, alanine, cysteine, asparticacid, glutamic acid, phenylalanine, glycine, histidine, isoleucine,lysine, methionine, asparagine, proline, glutamine, arginine, serine,threonine, valine, tryptophan, or tyrosine. For position 428, thesubstituting amino acid residues can be any amino acid residue otherthan methionine, including, but not limited to, alanine, cysteine,aspartic acid, glutamic acid, phenylalanine, glycine, histidine,isoleucine, lysine, leucine, asparagine, proline, glutamine, arginine,serine, threonine, valine, tryptophan, or tyrosine. Specificcombinations of suitable amino acid substitutions are identified inTable 1 of U.S. Pat. No. 7,217,797, which is incorporated herein byreference. Such mutations increase binding to FcRn, which protects theantibody from degradation and increases its half-life.

In yet other aspects, an anti-glyco-MUC1 antibody of antigen-bindingfragment of the disclosure has one or more amino acids inserted into oneor more of its hypervariable regions, for example as described in Jungand Pluckthun, 1997, Protein Engineering 10:9, 959-966; Yazaki et al.,2004, Protein Eng. Des Sel. 17(5):481-9. Epub 2004 Aug. 17; and U.S.Pat. App. No. 2007/0280931.

In yet other aspects, particularly useful for diagnostic applications,an anti-glyco-MUC1 antibody of antigen-binding fragment of thedisclosure is attached to a detectable moiety. Detectably moietiesinclude a radioactive moiety, a colorimetric molecule, a fluorescentmoiety, a chemiluminescent moiety, an antigen, an enzyme, a detectablebead (such as a magnetic or electrodense (e.g., gold) bead), or amolecule that binds to another molecule (e.g., biotin or streptavidin)).

Radioisotopes or radionuclides may include ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I,

Fluorescent labels may include rhodamine, lanthanide phosphors,fluorescein and its derivatives, fluorochrome, GFP (GFP for “GreenFluorescent Protein”), dansyl, umbelliferone, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine.

Enzymatic labels may include horseradish peroxidase, β galactosidase,luciferase, alkaline phosphatase, glucose-6-phosphate dehydrogenase(“G6PDH”), alpha-D-galactosidase, glucose oxydase, glucose amylase,carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenaseand peroxidase.

Chemiluminescent labels or chemiluminescers, such as isoluminol, luminoland the dioxetanes

Other detectable moieties include molecules such as biotin, digoxygeninor 5-bromodeoxyuridine.

In certain aspects, an anti-glyco-MUC1 antibody or antigen bindingfragment of the disclosure competes with GO2 or an antibody or antigenbinding fragment comprising heavy and light chain variable regions ofGO2 (SEQ ID NOS:3 and 4, respectively).

The competition can be assayed on cells that express the glyco-MUC1epitope bound by GO2 or on a glycosylated MUC1 peptide containing theepitope bound by GO2, e.g., the 60-mer peptide (VTSAPDTRPAPGSTAPPAHG)₃glycosylated with GalNAc-T2, GalNAc-T4, and GalNAc-T1 as described inU.S. Pat. No. 6,465,220. Cells that do not express the epitope orunglycosylated peptides can be used as controls.

Cells on which a competition assay can be carried out include but arenot limited to the breast cancer cell lines MCF7 or T47D and recombinantcells that are engineered to express the glyco-MUC1 epitope. In onenon-limiting example, CHO IdID cells, which lack the UDP-Gal/GalNAcepimerase and are deficient in GalNAc O-glycosylation andgalactosylation in the absence of exogenous addition of GalNAc and Gal,respectively, are engineered to express MUC1 and grown in the absence orpresence of GalNAc, the latter yielding cells expressing the Tnglycoform of MUC1 to which GO2 binds. Cells expressing theunglycosylated form of MUC1 can be used as a negative control.

Assays for competition include, but are not limited to, a radioactivematerial labeled immunoassay (RIA), an enzyme-linked immunosorbent assay(ELISA), a sandwich ELISA fluorescence activated cell sorting (FACS)assays and Biacore assays.

In conducting an antibody competition assay between a reference antibodyand a test antibody (irrespective of species or isotype), one may firstlabel the reference with a detectable label, such as a fluorophore,biotin or an enzymatic (or even radioactive) label to enable subsequentidentification. In this case, cells expressing glyco-MUC1 are incubatedwith unlabeled test antibody, labeled reference antibody is added, andthe intensity of the bound label is measured. If the test antibodycompetes with the labeled reference antibody by binding to anoverlapping epitope, the intensity will be decreased relative to acontrol reaction carried out without test antibody.

In a specific embodiment of this assay, the concentration of labeledreference antibody that yields 80% of maximal binding (“conc_(80%)%”)under the assay conditions (e.g., a specified density of cells) is firstdetermined, and a competition assay carried out with 10×conc_(80%) ofunlabeled test antibody and conc_(80%) of labeled reference antibody.

The inhibition can be expressed as an inhibition constant, or K_(i),which is calculated according to the following formula:

K_(i)=IC₅₀/(1+[reference Ab concentration]/K_(d)),

where IC₅₀ is the concentration of test antibody that yields a 50%reduction in binding of the reference antibody and K_(d) is thedissociation constant of the reference antibody, a measure of itsaffinity for glyco-MUC1. Antibodies that compete with anti-glyco-MUC1antibodies disclosed herein can have a K_(i) from 10 pM to 10 nM underassay conditions described herein.

In various embodiments, a test antibody is considered to compete with areference antibody if it decreases binding of the reference antibody byat least about 20% or more, for example, by at least about 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95% or even more, or by a percentageranging between any of the foregoing values, at a reference antibodyconcentration that is 80% of maximal binding under the specific assayconditions used, and a test antibody concentration that is 10-foldhigher than the reference antibody concentration.

In one example of a competition assay, the glycosylated MUC1 60-merpeptide is adhered onto a solid surface, e.g., a microwell plate, bycontacting the plate with a solution of the peptide (e.g., at aconcentration of 1 μg/mL in PBS over night at 4° C.). The plate iswashed (e.g., 0.1% Tween 20 in PBS) and blocked (e.g., in Superblock,Thermo Scientific, Rockford, Ill.). A mixture of sub-saturating amountof biotinylated GO2 (e.g., at a concentration of 80 ng/mL) and unlabeledGO2 (the “reference” antibody) or competing anti-glyco-MUC1 antibody(the “test” antibody) antibody in serial dilution (e.g., at aconcentration of 2.8 μg/mL, 8.3 μg/mL, or 25 μg/mL) in ELISA buffer(e.g., 1% BSA and 0.1% Tween 20 in PBS) is added to wells and plates areincubated for 1 hour with gentle shaking. The plate is washed, 1 μg/mLHRP-conjugated Streptavidin diluted in ELISA buffer is added to eachwell and the plates incubated for 1 hour. Plates are washed and boundantibodies were detected by addition of substrate (e.g., TMB, BiofxLaboratories Inc., Owings Mills, Md.). The reaction is terminated byaddition of stop buffer (e.g., Bio FX Stop Reagents, Biofx LaboratoriesInc., Owings Mills, Md.) and the absorbance is measured at 650 nm usingmicroplate reader (e.g., VERSAmax, Molecular Devices, Sunnyvale,Calif.).

Variations on this competition assay can also be used to testcompetition between GO2 and another anti-glyco-MUC1 antibody. Forexample, in certain aspects, the anti-glyco-MUC1 antibody is used as areference antibody and GO2 is used as a test antibody. Additionally,instead of glycosylated MUC1 60-mer peptide, membrane-bound glyco-MUC1expressed on cell surface (for example on the surface of one of the celltypes mentioned above) in culture can be used. Generally, about 10⁴ to10⁶ transfectants, e.g., about 10⁵ transfectants, are used. Otherformats for competition assays are known in the art and can be employed.

In various embodiments, an anti-glyco-MUC1 antibody of the disclosurereduces the binding of labeled GO2 by at least 40%, by at least 50%, byat least 60%, by at least 70%, by at least 80%, by at least 90%, or by apercentage ranging between any of the foregoing values (e.g., ananti-glyco-MUC1 antibody of the disclosure reduces the binding oflabeled GO2 by 50% to 70%) when the anti-glyco-MUC1 antibody is used ata concentration of 0.08 μg/mL, 0.4 μg/mL, 2 μg/mL, 10 μg/mL, 50 μg/mL,100 μg/mL or at a concentration ranging between any of the foregoingvalues (e.g., at a concentration ranging from 2 μg/mL to 10 μg/mL).

In other embodiments, GO2 reduces the binding of a labeledanti-glyco-MUC1 antibody of the disclosure by at least 40%, by at least50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%,or by a percentage ranging between any of the foregoing values (e.g.,GO2 reduces the binding of a labeled an anti-glyco-MUC1 antibody of thedisclosure by 50% to 70%) when GO2 is used at a concentration of 0.4μg/mL, 2 μg/mL, 10 μg/mL, 50 μg/mL, 250 μg/mL or at a concentrationranging between any of the foregoing values (e.g., at a concentrationranging from 2 μg/mL to 10 μg/mL).

In the foregoing assays, the GO2 antibody can be replaced by anyantibody or antigen-binding fragment comprising the CDRs or the heavyand light chain variable regions of GO2, such as a humanized or chimericcounterpart of GO2.

In certain aspects, an anti-glyco-MUC1 antibody or antigen-bindingfragment of the disclosure comprises heavy and/or light chain variablesequences (or encoded by the nucleotide sequences) set forth in Table 1.In other aspects, an anti-glyco-MUC1 antibody or antigen-bindingfragment of the disclosure comprises heavy and/or light chain CDRsequences (or encoded by the nucleotide sequences) set forth in Table 1.The framework sequences for such anti-glyco-MUC1 antibody andantigen-binding fragment can be the native murine framework sequences inTable 1 or can be non-native (e.g., humanized or human) frameworksequences.

In yet other aspects, the disclosure provides an anti-MUC1 antibody orantigen binding fragment having heavy and light chain variable regionshaving at least 95%, 98%, 99%, or 99.5% sequence identity of SEQ ID NOS:3 and 4, respectively.

In yet other aspects, an anti-glyco-MUC1 antibody or antigen-bindingfragment of the disclosure is a single-chain variable fragment (scFv).An exemplary scFv comprises the heavy chain variable fragment N-terminalto the light chain variable fragment. In some embodiments, the scFvheavy chain variable fragment and light chain variable fragment arecovalently bound to a linker sequence of 4-15 amino acids. The scFv canbe in the form of a bi-specific T-cell engager or within a chimericantigen receptor (CAR).

6.2 Anti-Glyco-MUC1 and Anti-CD3 Bispecific Antibodies

In some aspects, bispecific antibodies of the disclosure can comprise afirst antigen binding domain that specifically binds to CD3 (e.g., whichcomprises the CDRs or V_(H) and V_(L) set forth in Table 4), and asecond antigen binding domain that specifically binds to glyco-MUC1. Thesecond antigen binding domain may comprise, singly or in combination,the features described for the glyco-MUC1 antibodies hereinabove (e.g.,comprise a combination of CDRs identified in Tables 1-3, for exampleCDRs comprising the amino acid sequences of any of the CDR combinationsset forth in numbered embodiments 3 to 17, infra, or the V_(H) and V_(L)sequences identified in Table 1).

TABLE 4 SEQ Description Sequence ID NO: CD3 CDR-H1 TYAMN 34 (Kabat)CD3 CDR-H2 RIRSKYNNYATYYADSVKG 35 (Kabat) CD3 CDR-H3 HGNFGNSYVSWFAY 36(Kabat) CD3 CDR-L1 GSSTGAVTTSNYAN 37 (Kabat) CD3 CDR-L2 GTNKRAP 38(Kabat) CD3 CDR-L3 ALWYSNLWV 39 (Kabat) CD3 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPG 40KGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS CD3 VLQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKP 41GQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPED EAEYYCALWYSNLWVFGGGTKLTVLhIgG1 Fc DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV 42 regionVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSP MUC1 VL-DIVMSQSPSSLGVSVGEKVTMSCKSSQSLLYSTNQKNYQSLL 43 CL (RK)YSTNQKNYLAWYQQKPGQSPKLLIYWVSNRKSGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYRYPLTFGAGTKLELKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGECCD3 VH-CL EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPG 44KGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGECMUC1 VH- QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQRPE 45 CH1(EE)-QGLEWIGYFSPGNDDIHYNEKFEGKATLTADKSSSTAYMQLN Fc (hole, SLTSEDSAVYFCKRSYDKDFDCWGQGTTLTVSSASTKGPSVF P329G LALA)PLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKMUC1 VH- QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQRPE 46 CH1(EE)-QGLEWIGYFSPGNDDIHYNEKFEGKATLTADKSSSTAYMQLN CD3 VL-SLTSEDSAVYFCKRSYDKDFDCWGQGTTLTVSSASTKGPSVF CH1-FcPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVH (knob,TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK P329G LALA)VDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In some embodiments, the first antigen binding domain comprises a heavychain variable region comprising the heavy chain CDR-H1 of SEQ ID NO:34,the CDR-H2 of SEQ ID NO:35, and the CDR-H3 of SEQ ID NO:36; and a lightchain variable region comprising the light chain CDR-L1 of SEQ ID NO:37,the CDR-L2 of SEQ ID NO:38 and the CDR-L3 of SEQ ID NO:39.

In some embodiments, the second antigen binding domain comprises forexample CDRs comprising the amino acid sequences of any of the CDRcombinations set forth in numbered embodiments 3 to 17, for example (i)a heavy chain variable region comprising the heavy chain CDR-H1 of SEQID NO: 5, the CDR-H2 of SEQ ID NO: 6, and the CDR-H3 of SEQ ID NO: 7;and a light chain variable region comprising the light chain CDR (CDR-L)1 of SEQ ID NO: 8, the CDR-L2 of SEQ ID NO: 9 and the CDR-L3 of SEQ IDNO:10.

In a particular embodiment, the bispecific antibody comprises

(i) a first antigen binding domain that specifically binds to CD3 andcomprises a heavy chain variable region comprising a CDR-H1 comprisingthe amino acid sequence of SEQ ID NO:34, a CDR-H2 comprising the aminoacid sequence of SEQ ID NO:35, and a CDR-H3 comprising the amino acidsequence of SEQ ID NO:36; and a light chain variable region comprising aCDR-L1 comprising the amino acid sequence of SEQ ID NO:37, a CDR-L2comprising the amino acid sequence of SEQ ID NO:38, and a CDR-L3comprising the amino acid sequence of SEQ ID NO:39; and

(ii) a second antigen binding domain that specifically binds toglyco-MUC1 and comprises (i) a heavy chain variable region comprising aCDR-H1 comprising the amino acid sequence of SEQ ID NO:33, morepreferably a CDR-H1 comprising the amino acid sequence of SEQ ID NO:5, aCDR-H2 comprising the amino acid sequence of SEQ ID NO:29, morepreferably a CDR-H1 comprising the amino acid sequence of SEQ ID NO:6,and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:25, morepreferably a CDR-H3 comprising the amino acid sequence of SEQ ID NO:7;and a light chain variable region comprising a CDR-L1 comprising theamino acid sequence of of SEQ ID NO:8, a CDR-L2 comprising the aminoacid sequence of SEQ ID NO:9 and a CDR-L3 comprising the amino acidsequence of SEQ ID NO:31, more preferably a CDR-L3 comprising the aminoacid sequence of SEQ ID NO:10.

In some embodiments, the first antigen binding domain comprises a heavychain variable region sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:40and a light chain variable region sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO:41.

In some embodiments, the first antigen binding domain comprises theheavy chain variable region sequence of SEQ ID NO:40 and the light chainvariable region sequence of SEQ ID NO:41.

In some embodiments, the second antigen binding domain comprises a heavychain variable region sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:3 anda light chain variable region sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ IDNO:4.

In some embodiments, the second antigen binding domain comprises theheavy chain variable region sequence of SEQ ID NO:3 and the light chainvariable region sequence of SEQ ID NO:4.

In some embodiments, the first and/or the second antigen binding domainis a Fab molecule. In some embodiments, the first antigen binding domainis a crossover Fab molecule wherein either the variable or the constantregions of the Fab light chain and the Fab heavy chain are exchanged. Insuch embodiments, the second antigen binding domain preferably is aconventional Fab molecule.

In some embodiments wherein the first and the second antigen bindingdomain of the bispecific antibody are both Fab molecules, and in one ofthe antigen binding domains (particularly the first antigen bindingdomain) the variable domains VL and VH of the Fab light chain and theFab heavy chain are replaced by each other,

i) in the constant domain CL of the first antigen binding domain theamino acid at position 124 is substituted by a positively charged aminoacid (numbering according to Kabat), and wherein in the constant domainCH1 of the first antigen binding domain the amino acid at position 147or the amino acid at position 213 is substituted by a negatively chargedamino acid (numbering according to Kabat EU index); or

ii) in the constant domain CL of the second antigen binding domain theamino acid at position 124 is substituted by a positively charged aminoacid (numbering according to Kabat), and wherein in the constant domainCH1 of the second antigen binding domain the amino acid at position 147or the amino acid at position 213 is substituted by a negatively chargedamino acid (numbering according to Kabat EU index).

The bispecific antibody does not comprise both modifications mentionedunder i) and ii). The constant domains CL and CH1 of the antigen bindingdomain having the VH/VL exchange are not replaced by each other (i.e.,they remain unexchanged).

In a more specific embodiment,

i) in the constant domain CL of the first antigen binding domain theamino acid at position 124 is substituted independently by lysine (K),arginine (R) or histidine (H) (numbering according to Kabat), and in theconstant domain CH1 of the first antigen binding domain the amino acidat position 147 or the amino acid at position 213 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index); or

ii) in the constant domain CL of the second antigen binding domain theamino acid at position 124 is substituted independently by lysine (K),arginine (R) or histidine (H) (numbering according to Kabat), and in theconstant domain CH1 of the second antigen binding domain the amino acidat position 147 or the amino acid at position 213 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).

In one such embodiment, in the constant domain CL of the second antigenbinding domain the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the secondantigen binding domain the amino acid at position 147 or the amino acidat position 213 is substituted independently by glutamic acid (E), oraspartic acid (D) (numbering according to Kabat EU index).

In a further embodiment, in the constant domain CL of the second antigenbinding domain the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the secondantigen binding domain the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).

In a particular embodiment, in the constant domain CL of the secondantigen binding domain the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) and the amino acid at position 123 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the secondantigen binding domain the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index).

In a more particular embodiment, in the constant domain CL of the secondantigen binding domain the amino acid at position 124 is substituted bylysine (K) (numbering according to Kabat) and the amino acid at position123 is substituted by lysine (K) (numbering according to Kabat), and inthe constant domain CH1 of the second antigen binding domain the aminoacid at position 147 is substituted by glutamic acid (E) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted by glutamic acid (E) (numbering according to Kabat EUindex).

In an even more particular embodiment, in the constant domain CL of thesecond antigen binding domain the amino acid at position 124 issubstituted by lysine (K) (numbering according to Kabat) and the aminoacid at position 123 is substituted by arginine (R) (numbering accordingto Kabat), and in the constant domain CH1 of the second antigen bindingdomain the amino acid at position 147 is substituted by glutamic acid(E) (numbering according to Kabat EU index) and the amino acid atposition 213 is substituted by glutamic acid (E) (numbering according toKabat EU index).

In particular embodiments, if amino acid substitutions according to theabove embodiments are made in the constant domain CL and the constantdomain CH1 of the second antigen binding domain, the constant domain CLof the second antigen binding domain is of kappa isotype.

In some embodiments, the first and the second antigen binding domain arefused to each other, optionally via a peptide linker.

In some embodiments, the first and the second antigen binding domain areeach a Fab molecule and either (i) the second antigen binding domain isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the first antigen binding domain, or (ii) the firstantigen binding domain is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second antigen bindingdomain.

In some embodiments, the bispecific antibody provides monovalent bindingto CD3.

In particular embodiments, the bispecific antibody comprises a singleantigen binding domain that specifically binds to CD3, and two antigenbinding domains that specifically bind to glyco-MUC1. Thus, in someembodiments, the bispecific antibody comprises a third antigen bindingdomain that specifically binds to glyco-MUC1. In some embodiments, thethird antigen moiety is identical to the first antigen binding domain(e.g. is also a Fab molecule and comprises the same amino acidsequences).

In particular embodiments, the bispecific antibody further comprises anFc domain composed of a first and a second subunit. In one embodiment,the Fc domain is an IgG Fc domain. In a particular embodiment, the Fcdomain is an IgG₁ Fc domain. In another embodiment the Fc domain is anIgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG₄Fc domain comprising an amino acid substitution at position S228 (KabatEU index numbering), particularly the amino acid substitution S228P. Ina further particular embodiment, the Fc domain is a human Fc domain. Inan even more particular embodiment, the Fc domain is a human IgG₁ Fcdomain. An exemplary sequence of a human IgG₁ Fc region is given in SEQID NO: 42.

In some embodiments wherein the first, the second and, where present,the third antigen binding domain are each a Fab molecule, (a) either (i)the second antigen binding domain is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the firstantigen binding domain and the first antigen binding domain is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the firstsubunit of the Fc domain, or (ii) the first antigen binding domain isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the second antigen binding domain and the secondantigen binding domain is fused at the C-terminus of the Fab heavy chainto the N-terminus of the first subunit of the Fc domain; and (b) thethird antigen binding domain, where present, is fused at the C-terminusof the Fab heavy chain to the N-terminus of the second subunit of the Fcdomain.

In particular embodiments, the Fc domain comprises a modificationpromoting the association of the first and the second subunit of the Fcdomain, for example, as described in Section 6.1.

In some embodiments, the Fc domain comprises one or more amino acidsubstitutions that reduces binding to an Fc receptor and/or effectorfunction, for example as described in Section 6.1.

In a particular embodiment the bispecific antibody comprises

(i) a first antigen binding domain that specifically binds to CD3,wherein the first antigen binding domain is a crossover Fab moleculewherein either the variable or the constant regions, particularly thevariable regions, of the Fab light chain and the Fab heavy chain areexchanged;

(ii) a second and a third antigen binding domain that specifically bindto glyco-MUC1, comprising a heavy chain variable region comprising theheavy chain CDR-H1 of SEQ ID NO: 5, the CDR-H2 of SEQ ID NO: 6, and theCDR-H3 of SEQ ID NO: 7; and a light chain variable region comprising thelight chain CDR-L1 of SEQ ID NO: 8, the CDR-L2 of SEQ ID NO: 9 and theCDR-L3 of SEQ ID NO:10, wherein the second and third antigen bindingdomain are each a Fab molecule, particularly a conventional Fabmolecule;

(iii) an Fc domain composed of a first and a second subunit capable ofstable association,

wherein the second antigen binding domain is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst antigen binding domain, and the first antigen binding domain isfused at the C-terminus of the Fab heavy chain to the N-terminus of thefirst subunit of the Fc domain, and wherein the third antigen bindingdomain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the second subunit of the Fc domain.

In one embodiment the first antigen binding domain comprises a heavychain variable region comprising the heavy chain CDR-H1 of SEQ ID NO:34,the CDR-H2 of SEQ ID NO:35, and the CDR-H3 of SEQ ID NO:36; and a lightchain variable region comprising the light chain CDR-L1 of SEQ ID NO:37,the CDR-L2 of SEQ ID NO:38 and the CDR-L3 of SEQ ID NO:39.

In one embodiment, the first antigen binding domain comprises a heavychain variable region sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:40and a light chain variable region sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO:41.

In one embodiment, the first antigen binding domain comprises the heavychain variable region sequence of SEQ ID NO:40 and the light chainvariable region sequence of SEQ ID NO:41.

In one embodiment, the second and third antigen binding domain comprisea heavy chain variable region sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ IDNO:3 and a light chain variable region sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO:4. Preferably, the antigen binding domain comprises CDRscomprising the amino acid sequences of any of the CDR combinations setforth in numbered embodiments 3 to 17. In one embodiment, the second andthird antigen binding domains comprise the heavy chain variable regionof SEQ ID NO:3 and the light chain variable region of SEQ ID NO:4.

The Fc domain according to the above embodiments may incorporate, singlyor in combination, all of the features described hereinabove in relationto Fc domains.

In some embodiments, the antigen binding domains and the Fc region arefused to each other by peptide linkers, for example by peptide linkersas in SEQ ID NO:45 and SEQ ID NO:46.

In one embodiment, in the constant domain CL of the second and the thirdFab molecule under (ii) the amino acid at position 124 is substituted bylysine (K) (numbering according to Kabat) and the amino acid at position123 is substituted by lysine (K) or arginine (R), particularly byarginine (R) (numbering according to Kabat), and in the constant domainCH1 of the second and the third Fab molecule under (ii) the amino acidat position 147 is substituted by glutamic acid (E) (numbering accordingto Kabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index).

In one embodiment, the bispecific antibody comprises a polypeptidecomprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO:43 (and preferablycomprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, aCDR-L2 comprising the amino acid sequence of SEQ ID NO:9, and a CDR-L3comprising the amino acid sequence of SEQ ID NO:31), a polypeptidecomprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to the sequence of SEQ ID NO:44 (and preferablycomprises the CD3 heavy and light chain CDR sequences set forth in Table4), a polypeptide comprising a sequence that is at least 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:45(and preferably comprises a CDR-H1 comprising the amino acid sequence ofSEQ ID NO:33, a CDR-H2 comprising the amino acid sequence of SEQ IDNO:29, a CDR-H3 comprising the amino acid sequence of SEQ ID NO:25), anda polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:46 (andpreferably comprises a CDR-H1 comprising the amino acid sequence of SEQID NO:33, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:29, aCDR-H3 comprising the amino acid sequence of SEQ ID NO:25, a CDR-L1comprising the amino acid sequence of SEQ ID NO:37, a CDR-L2 comprisingthe amino acid sequence of SEQ ID NO:38, and a CDR-L3 comprising theamino acid sequence of SEQ ID NO:39).

In one embodiment, the bispecific antibody comprises a polypeptide(particularly two polypeptides) comprising the sequence of SEQ ID NO:43,a polypeptide comprising the sequence of SEQ ID NO:44, a polypeptidecomprising the sequence of SEQ ID NO:45, and a polypeptide comprisingthe sequence of SEQ ID NO:46.

6.3 Antibody-Drug Conjugates

Another aspect of the disclosure concerns antibody drug conjugates(ADCs) including the anti-glyco-MUC1 antibodies and antigen-bindingfragments of the disclosure. The ADCs generally comprise ananti-glyco-MUC1 antibody and/or binding fragment as described hereinhaving one or more cytotoxic and/or cytostatic agents linked thereto byway of one or more linkers. In specific embodiments, the ADCs arecompounds according to structural formula (I):

[D-L-XY]_(n)-Ab

or salts thereof, where each “D” represents, independently of theothers, a cytotoxic and/or cytostatic agent (“drug”); each “L”represents, independently of the others, a linker; “Ab” represents ananti-glyco-MUC1 antigen binding domain, such as an anti-glyco-MUC1antibody or binding fragment described herein; each “XY” represents alinkage formed between a functional group R^(x) on the linker and a“complementary” functional group R^(y) on the antibody, and n representsthe number of drugs linked to, or drug-to-antibody ratio (DAR), of theADC.

Specific embodiments of the various antibodies (Ab) that can comprisethe ADCs include the various embodiments of anti-glyco-MUC1 antibodiesand/or binding fragments described above.

In some specific embodiments of the ADCs and/or salts of structuralformula (I), each D is the same and/or each L is the same.

Specific embodiments of cytotoxic and/or cytostatic agents (D) andlinkers (L) that can comprise the anti-glyco-MUC1 ADCs of thedisclosure, as well as the number of cytotoxic and/or cytostatic agentslinked to the ADCs, are described in more detail below.

6.3.1. Cytotoxic and/or Cytostatic Agents

The cytotoxic and/or cytostatic agents may be any agents known toinhibit the growth and/or replication of and/or kill cells, and inparticular cancer and/or tumor cells. Numerous agents having cytotoxicand/or cytostatic properties are known in the literature. Non-limitingexamples of classes of cytotoxic and/or cytostatic agents include, byway of example and not limitation, radionuclides, alkylating agents,topoisomerase I inhibitors, topoisomerase II inhibitors, DNAintercalating agents (e.g., groove binding agents such as minor groovebinders), RNA/DNA antimetabolites, cell cycle modulators, kinaseinhibitors, protein synthesis inhibitors, histone deacetylaseinhibitors, mitochondria inhibitors, and antimitotic agents.

Specific non-limiting examples of agents within certain of these variousclasses are provided below.

Alkylatinq Agents: asaley ((L-Leucine,N-[N-acetyl-4-[bis-(2-chloroethyl)amino]-DL-phenylalanyl]-, ethylester;NSC 167780; CAS Registry No. 3577897)); AZQ((1,4-cyclohexadiene-1,4-dicarbamic acid,2,5-bis(1-aziridinyl)-3,6-dioxo-, diethyl ester; NSC 182986; CASRegistry No. 57998682)); BCNU ((N,N′-Bis(2-chloroethyl)-N-nitrosourea;NSC 409962; CAS Registry No. 154938)); busulfan (1,4-butanedioldimethanesulfonate; NSC 750; CAS Registry No. 55981);(carboxyphthalato)platinum (NSC 27164; CAS Registry No. 65296813); CBDCA((cis-(1,1-cyclobutanedicarboxylato)diammineplatinum(II)); NSC 241240;CAS Registry No. 41575944)); CCNU((N-(2-chloroethyl)-N′-cyclohexyl-N-nitrosourea; NSC 79037; CAS RegistryNo. 13010474)); CHIP (iproplatin; NSC 256927); chlorambucil (NSC 3088;CAS Registry No. 305033); chlorozotocin ((2-[[[(2-chloroethyl)nitrosoamino]carbonyl]amino]-2-deoxy-D-glucopyranose; NSC 178248; CASRegistry No. 54749905)); cis-platinum (cisplatin; NSC 119875; CASRegistry No. 15663271); clomesone (NSC 338947; CAS Registry No.88343720); cyanomorpholinodoxorubicin (NCS 357704; CAS Registry No.88254073); cyclodisone (NSC 348948; CAS Registry No. 99591738);dianhydrogalactitol (5,6-diepoxydulcitol; NSC 132313; CAS Registry No.23261203); fluorodopan((5-[(2-chloroethyl)-(2-fluoroethyl)amino]-6-methyl-uracil; NSC 73754;CAS Registry No. 834913); hepsulfam (NSC 329680; CAS Registry No.96892578); hycanthone (NSC 142982; CAS Registry No. 23255938); melphalan(NSC 8806; CAS Registry No. 3223072); methyl CCNU((1-(2-chloroethyl)-3-(trans-4-methylcyclohexane)-1-nitrosourea; NSC95441; 13909096); mitomycin C (NSC 26980; CAS Registry No. 50077);mitozolamide (NSC 353451; CAS Registry No. 85622953); nitrogen mustard((bis(2-chloroethyl)methylamine hydrochloride; NSC 762; CAS Registry No.55867); PCNU((1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea; NSC 95466;CAS Registry No. 13909029)); piperazine alkylator((1-(2-chloroethyl)-4-(3-chloropropyl)-piperazine dihydrochloride; NSC344007)); piperazinedione (NSC 135758; CAS Registry No. 41109802);pipobroman ((N,N-bis(3-bromopropionyl) piperazine; NSC 25154; CASRegistry No. 54911)); porfiromycin (N-methylmitomycin C; NSC 56410; CASRegistry No. 801525); spirohydantoin mustard (NSC 172112; CAS RegistryNo. 56605164); teroxirone (triglycidylisocyanurate; NSC 296934; CASRegistry No. 2451629); tetraplatin (NSC 363812; CAS Registry No.62816982); thio-tepa (N,N′,N″-tri-1,2-ethanediylthio phosphoramide; NSC6396; CAS Registry No. 52244); triethylenemelamine (NSC 9706; CASRegistry No. 51183); uracil nitrogen mustard (desmethyldopan; NSC 34462;CAS Registry No. 66751); Yoshi-864 ((bis(3-mesyloxy propyl)aminehydrochloride; NSC 102627; CAS Registry No. 3458228).

Topoisomerase I Inhibitors: camptothecin (NSC 94600; CAS Registry No.7689-03-4); various camptothecin derivatives and analogs (for example,NSC 100880, NSC 603071, NSC 107124, NSC 643833, NSC 629971, NSC 295500,NSC 249910, NSC 606985, NSC 74028, NSC 176323, NSC 295501, NSC 606172,NSC 606173, NSC 610458, NSC 618939, NSC 610457, NSC 610459, NSC 606499,NSC 610456, NSC 364830, and NSC 606497); morpholinisoxorubicin (NSC354646; CAS Registry No. 89196043); SN-38 (NSC 673596; CAS Registry No.86639-52-3).

Topoisomerase II Inhibitors: doxorubicin (NSC 123127; CAS Registry No.25316409); amonafide (benzisoquinolinedione; NSC 308847; CAS RegistryNo. 69408817); m-AMSA((4′-(9-acridinylamino)-3′-methoxymethanesulfonanilide; NSC 249992; CASRegistry No. 51264143)); anthrapyrazole derivative ((NSC 355644);etoposide (VP-16; NSC 141540; CAS Registry No. 33419420);pyrazoloacridine ((pyrazolo[3,4,5-kl]acridine-2(6H)-propanamine,9-methoxy-N,N-dimethyl-5-nitro-, monomethanesulfonate; NSC 366140; CASRegistry No. 99009219); bisantrene hydrochloride (NSC 337766; CASRegistry No. 71439684); daunorubicin (NSC 821151; CAS Registry No.23541506); deoxydoxorubicin (NSC 267469; CAS Registry No. 63950061);mitoxantrone (NSC 301739; CAS Registry No. 70476823); menogaril (NSC269148; CAS Registry No. 71628961); N,N-dibenzyl daunomycin (NSC 268242;CAS Registry No. 70878512); oxanthrazole (NSC 349174; CAS Registry No.105118125); rubidazone (NSC 164011; CAS Registry No. 36508711);teniposide (VM-26; NSC 122819; CAS Registry No. 29767202).

DNA Intercalating Agents: anthramycin (CAS Registry No. 4803274);chicamycin A (CAS Registry No. 89675376); tomaymycin (CAS Registry No.35050556); DC-81 (CAS Registry No. 81307246); sibiromycin (CAS RegistryNo. 12684332); pyrrolobenzodiazepine derivative (CAS Registry No.945490095); SGD-1882((S)-2-(4-aminophenyl)-7-methoxy-8-(3-4(S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propox-y)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one);SG2000 (SJG-136;(11aS,11a′S)-8,8′-(propane-1,3-diylbis(oxy))bis(7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one);NSC 694501; CAS Registry No. 232931576).

RNA/DNA Antimetabolites: L-alanosine (NSC 153353; CAS Registry No.59163416); 5-azacytidine (NSC 102816; CAS Registry No. 320672);5-fluorouracil (NSC 19893; CAS Registry No. 51218); acivicin (NSC163501; CAS Registry No. 42228922); aminopterin derivativeN-[2-chloro-5-[[(2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino]benzoyl-]L-asparticacid (NSC 132483); aminopterin derivativeN-[4-[[(2,4-diamino-5-ethyl-6-quinazolinyhmethyl]amino]benzoyl]L-asparti-cacid (NSC 184692); aminopterin derivativeN-[2-chloro-4-[[(2,4-diamino-6-pteridinyl)methyl]amino]benzoyl]L-asparticacid monohydrate (NSC 134033); an antifo((N^(α)-(4-amino-4-deoxypteroyl)-N⁷-hemiphthaloyl-L-ornithin-e; NSC623017)); Bakers soluble antifol (NSC 139105; CAS Registry No.41191042); dichlorallyl lawsone((2-(3,3-dichloroallyl)-3-hydroxy-1,4-naphthoquinone; NSC 126771; CASRegistry No. 36417160); brequinar (NSC 368390; CAS Registry No.96201886); ftorafur ((pro-drug; 5-fluoro-1-(tetrahydro-2-furyl)-uracil;NSC 148958; CAS Registry No. 37076689); 5,6-dihydro-5-azacytidine (NSC264880; CAS Registry No. 62402317); methotrexate (NSC 740; CAS RegistryNo. 59052); methotrexate derivative(N-[[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]-1-naphthalenyl]car-bonyl]L-glutamicacid; NSC 174121); PALA ((N-(phosphonoacetyl)-L-aspartate; NSC 224131;CAS Registry No. 603425565); pyrazofurin (NSC 143095; CAS Registry No.30868305); trimetrexate (NSC 352122; CAS Registry No. 82952645).

DNA Antimetabolites: 3-HP (NSC 95678; CAS Registry No. 3814797);2′-deoxy-5-fluorouridine (NSC 27640; CAS Registry No. 50919); 5-HP (NSC107392; CAS Registry No. 19494894); α-TGDR (α-2′-deoxy-6-thioguanosine;NSC 71851 CAS Registry No. 2133815); aphidicolin glycinate (NSC 303812;CAS Registry No. 92802822); ara C (cytosine arabinoside; NSC 63878; CASRegistry No. 69749); 5-aza-2′-deoxycytidine (NSC 127716; CAS RegistryNo. 2353335); β-TGDR (β-2′-deoxy-6-thioguanosine; NSC 71261; CASRegistry No. 789617); cyclocytidine (NSC 145668; CAS Registry No.10212256); guanazole (NSC 1895; CAS Registry No. 1455772); hydroxyurea(NSC 32065; CAS Registry No. 127071); inosine glycodialdehyde (NSC118994; CAS Registry No. 23590990); macbecin II (NSC 330500; CASRegistry No. 73341738); pyrazoloimidazole (NSC 51143; CAS Registry No.6714290); thioguanine (NSC 752; CAS Registry No. 154427); thiopurine(NSC 755; CAS Registry No. 50442).

Cell Cycle Modulators: silibinin (CAS Registry No. 22888-70-6);epigallocatechin gallate (EGCG; CAS Registry No. 989515); procyanidinderivatives (e.g., procyanidin A2 [CAS Registry No. 103883030],procyanidin B1 [CAS Registry No. 20315257], procyanidin B4 [CAS RegistryNo. 29106512], are catannin B1 [CAS Registry No. 79763283]); isoflavones(e.g., genistein [4%5,7-trihydroxyisoflavone; CAS Registry No. 446720],daidzein [4′,7-dihydroxyisoflavone, CAS Registry No. 486668];indole-3-carbinol (CAS Registry No. 700061); quercetin (NSC 9219; CASRegistry No. 117395); estramustine (NSC 89201; CAS Registry No.2998574); nocodazole (CAS Registry No. 31430189); podophyllotoxin (CASRegistry No. 518285); vinorelbine tartrate (NSC 608210; CAS Registry No.125317397); cryptophycin (NSC 667642; CAS Registry No. 124689652).

Kinase Inhibitors: afatinib (CAS Registry No. 850140726); axitinib (CASRegistry No. 319460850); ARRY-438162 (binimetinib) (CAS Registry No.606143899); bosutinib (CAS Registry No. 380843754); cabozantinib (CASRegistry No. 1140909483); ceritinib (CAS Registry No. 1032900256);crizotinib (CAS Registry No. 877399525); dabrafenib (CAS Registry No.1195765457); dasatinib (NSC 732517; CAS Registry No. 302962498);erlotinib (NSC 718781; CAS Registry No. 183319699); everolimus (NSC733504; CAS Registry No. 159351696); fostamatinib (NSC 745942; CASRegistry No. 901119355); gefitinib (NSC 715055; CAS Registry No.184475352); ibrutinib (CAS Registry No. 936563961); imatinib (NSC716051; CAS Registry No. 220127571); lapatinib (CAS Registry No.388082788); lenvatinib (CAS Registry No. 857890392); mubritinib (CAS366017096); nilotinib (CAS Registry No. 923288953); nintedanib (CASRegistry No. 656247175); palbociclib (CAS Registry No. 571190302);pazopanib (NSC 737754; CAS Registry No. 635702646); pegaptanib (CASRegistry No. 222716861); ponatinib (CAS Registry No. 1114544318);rapamycin (NSC 226080; CAS Registry No. 53123889); regorafenib (CASRegistry No. 755037037); AP 23573 (ridaforolimus) (CAS Registry No.572924540); INCB018424 (ruxolitinib) (CAS Registry No. 1092939177);ARRY-142886 (selumetinib) (NSC 741078; CAS Registry No. 606143-52-6);sirolimus (NSC 226080; CAS Registry No. 53123889); sorafenib (NSC724772; CAS Registry No. 475207591); sunitinib (NSC 736511; CAS RegistryNo. 341031547); tofacitinib (CAS Registry No. 477600752); temsirolimus(NSC 683864; CAS Registry No. 163635043); trametinib (CAS Registry No.871700173); vandetanib (CAS Registry No. 443913733); vemurafenib (CASRegistry No. 918504651); SU6656 (CAS Registry No. 330161870); CEP-701(lesaurtinib) (CAS Registry No. 111358884); XL019 (CAS Registry No.945755566); PD-325901 (CAS Registry No. 391210109); PD-98059 (CASRegistry No. 167869218); ATP-competitive TORC1/TORC2 inhibitorsincluding PI-103 (CAS Registry No. 371935749), PP242 (CAS Registry No.1092351671), PP30 (CAS Registry No. 1092788094), Torin 1 (CAS RegistryNo. 1222998368), LY294002 (CAS Registry No. 154447366), XL-147 (CASRegistry No. 934526893), CAL-120 (CAS Registry No. 870281348), ETP-45658(CAS Registry No. 1198357797), PX 866 (CAS Registry No. 502632668),GDC-0941 (CAS Registry No. 957054307), BGT226 (CAS Registry No.1245537681), BEZ235 (CAS Registry No. 915019657), XL-765 (CAS RegistryNo. 934493762).

Protein Synthesis Inhibitors: acriflavine (CAS Registry No. 65589700);amikacin (NSC 177001; CAS Registry No. 39831555); arbekacin (CASRegistry No. 51025855); astromicin (CAS Registry No. 55779061);azithromycin (NSC 643732; CAS Registry No. 83905015); bekanamycin (CASRegistry No. 4696768); chlortetracycline (NSC 13252; CAS Registry No.64722); clarithromycin (NSC 643733; CAS Registry No. 81103119);clindamycin (CAS Registry No. 18323449); clomocycline (CAS Registry No.1181540); cycloheximide (CAS Registry No. 66819); dactinomycin (NSC3053; CAS Registry No. 50760); dalfopristin (CAS Registry No.112362502); demeclocycline (CAS Registry No. 127333); dibekacin (CASRegistry No. 34493986); dihydrostreptomycin (CAS Registry No. 128461);dirithromycin (CAS Registry No. 62013041); doxycycline (CAS Registry No.17086281); emetine (NSC 33669; CAS Registry No. 483181); erythromycin(NSC 55929; CAS Registry No. 114078); flurithromycin (CAS Registry No.83664208); framycetin (neomycin B; CAS Registry No. 119040); gentamycin(NSC 82261; CAS Registry No. 1403663); glycylcyclines, such astigecycline (CAS Registry No. 220620097); hygromycin B (CAS Registry No.31282049); isepamicin (CAS Registry No. 67814760); josamycin (NSC122223; CAS Registry No. 16846245); kanamycin (CAS Registry No.8063078); ketolides such as telithromycin (CAS Registry No. 191114484),cethromycin (CAS Registry No. 205110481), and solithromycin (CASRegistry No. 760981837); lincomycin (CAS Registry No. 154212);lymecycline (CAS Registry No. 992212); meclocycline (NSC 78502; CASRegistry No. 2013583); metacycline (rondomycin; NSC 356463; CAS RegistryNo. 914001); midecamycin (CAS Registry No. 35457808); minocycline (NSC141993; CAS Registry No. 10118908); miocamycin (CAS Registry No.55881077); neomycin (CAS Registry No. 119040); netilmicin (CAS RegistryNo. 56391561); oleandomycin (CAS Registry No. 3922905); oxazolidinones,such as eperezolid (CAS Registry No. 165800044), linezolid (CAS RegistryNo. 165800033), posizolid (CAS Registry No. 252260029), radezolid (CASRegistry No. 869884786), ranbezolid (CAS Registry No. 392659380),sutezolid (CAS Registry No. 168828588), tedizolid (CAS Registry No.856867555); oxytetracycline (NSC 9169; CAS Registry No. 2058460);paromomycin (CAS Registry No. 7542372); penimepicycline (CAS RegistryNo. 4599604); peptidyl transferase inhibitors, e.g., chloramphenicol(NSC 3069; CAS Registry No. 56757) and derivatives such as azidamfenicol(CAS Registry No. 13838089), florfenicol (CAS Registry No. 73231342),and thiamphenicol (CAS Registry No. 15318453), and pleuromutilins suchas retapamulin (CAS Registry No. 224452668), tiamulin (CAS Registry No.55297955), valnemulin (CAS Registry No. 101312929); pirlimycin (CASRegistry No. 79548735); puromycin (NSC 3055; CAS Registry No. 53792);quinupristin (CAS Registry No. 120138503); ribostamycin (CAS RegistryNo. 53797356); rokitamycin (CAS Registry No. 74014510); rolitetracycline(CAS Registry No. 751973); roxithromycin (CAS Registry No. 80214831);sisomicin (CAS Registry No. 32385118); spectinomycin (CAS Registry No.1695778); spiramycin (CAS Registry No. 8025818); streptogramins such aspristinamycin (CAS Registry No. 270076603), quinupristin/dalfopristin(CAS Registry No. 126602899), and virginiamycin (CAS Registry No.11006761); streptomycin (CAS Registry No. 57921); tetracycline (NSC108579; CAS Registry No. 60548); tobramycin (CAS Registry No. 32986564);troleandomycin (CAS Registry No. 2751099); tylosin (CAS Registry No.1401690); verdamicin (CAS Registry No. 49863481).

Histone Deacetylase Inhibitors: abexinostat (CAS Registry No.783355602); belinostat (NSC 726630; CAS Registry No. 414864009);chidamide (CAS Registry No. 743420022); entinostat (CAS Registry No.209783802); givinostat (CAS Registry No. 732302997); mocetinostat (CASRegistry No. 726169739); panobinostat (CAS Registry No. 404950807);quisinostat (CAS Registry No. 875320299); resminostat (CAS Registry No.864814880); romidepsin (CAS Registry No. 128517077); sulforaphane (CASRegistry No. 4478937); thioureidobutyronitrile (Kevetrin™; CAS RegistryNo. 6659890); valproic acid (NSC 93819; CAS Registry No. 99661);vorinostat (NSC 701852; CAS Registry No. 149647789); ACY-1215(rocilinostat; CAS Registry No. 1316214524); CUDC-101 (CAS Registry No.1012054599); CHR-2845 (tefinostat; CAS Registry No. 914382608); CHR-3996(CAS Registry No. 1235859138); 4SC-202 (CAS Registry No. 910462430);CG200745 (CAS Registry No. 936221339); SB939 (pracinostat; CAS RegistryNo. 929016966).

Mitochondria Inhibitors: pancratistatin (NSC 349156; CAS Registry No.96281311); rhodamine-123 (CAS Registry No. 63669709); edelfosine (NSC324368; CAS Registry No. 70641519); d-alpha-tocopherol succinate (NSC173849; CAS Registry No. 4345033); compound 11β (CAS Registry No.865070377); aspirin (NSC 406186; CAS Registry No. 50782); ellipticine(CAS Registry No. 519233); berberine (CAS Registry No. 633658);cerulenin (CAS Registry No. 17397896); GX015-070 (Obatoclax®; 1H-Indole,2-(2-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-;NSC 729280; CAS Registry No. 803712676); celastrol (tripterine; CASRegistry No. 34157830); metformin (NSC 91485; CAS Registry No. 1115704);Brilliant green (NSC 5011; CAS Registry No. 633034); ME-344 (CASRegistry No. 1374524556).

Antimitotic Agents: allocolchicine (NSC 406042); auristatins, such asMMAE (monomethyl auristatin E; CAS Registry No. 474645-27-7) and MMAF(monomethyl auristatin F; CAS Registry No. 745017-94-1; halichondrin B(NSC 609395); colchicine (NSC 757; CAS Registry No. 64868); cholchicinederivative (N-benzoyl-deacetyl benzamide; NSC 33410; CAS Registry No.63989753); dolastatin 10 (NSC 376128; CAS Registry No 110417-88-4);maytansine (NSC 153858; CAS Registry No. 35846-53-8); rhozoxin (NSC332598; CAS Registry No. 90996546); taxol (NSC 125973; CAS Registry No.33069624); taxol derivative ((2′-N-[3-(dimethylamino)propyl]glutaramatetaxol; NSC 608832); thiocolchicine (3-demethylthiocolchicine; NSC361792); trityl cysteine (NSC 49842; CAS Registry No. 2799077);vinblastine sulfate (NSC 49842; CAS Registry No. 143679); vincristinesulfate (NSC 67574; CAS Registry No. 2068782).

Any of these agents that include or that may be modified to include asite of attachment to an antibody may be included in the ADCs disclosedherein.

In a specific embodiment, the cytotoxic and/or cytostatic agent is anantimitotic agent.

In another specific embodiment, the cytotoxic and/or cytostatic agent isan auristatin, for example, monomethyl auristatin E (“MMAE”) ormonomethyl auristatin F (“MMAF”).

6.3.2. Linkers

In the anti-glyco-MUC1 ADCs of the disclosure, the cytotoxic and/orcytostatic agents are linked to the antibody by way of linkers. Thelinker linking a cytotoxic and/or cytostatic agent to the antibody of anADC may be short, long, hydrophobic, hydrophilic, flexible or rigid, ormay be composed of segments that each independently have one or more ofthe above-mentioned properties such that the linker may include segmentshaving different properties. The linkers may be polyvalent such thatthey covalently link more than one agent to a single site on theantibody, or monovalent such that covalently they link a single agent toa single site on the antibody.

As will be appreciated by skilled artisans, the linkers link cytotoxicand/or cytostatic agents to the antibody by forming a covalent linkageto the cytotoxic and/or cytostatic agent at one location and a covalentlinkage to antibody at another. The covalent linkages are formed byreaction between functional groups on the linker and functional groupson the agents and antibody. As used herein, the expression “linker” isintended to include (i) unconjugated forms of the linker that include afunctional group capable of covalently linking the linker to a cytotoxicand/or cytostatic agent and a functional group capable of covalentlylinking the linker to an antibody; (ii) partially conjugated forms ofthe linker that includes a functional group capable of covalentlylinking the linker to an antibody and that is covalently linked to acytotoxic and/or cytostatic agent, or vice versa; and (iii) fullyconjugated forms of the linker that is covalently linked to both acytotoxic and/or cytostatic agent and an antibody. In some specificembodiments of linkers and anti-glyco-MUC1 ADCs of the disclosure, aswell as synthons used to conjugate linker-agents to antibodies, moietiescomprising the functional groups on the linker and covalent linkagesformed between the linker and antibody are specifically illustrated asR_(x) and XY, respectively.

The linkers are preferably, but need not be, chemically stable toconditions outside the cell, and may be designed to cleave, immolateand/or otherwise specifically degrade inside the cell. Alternatively,linkers that are not designed to specifically cleave or degrade insidethe cell may be used. Choice of stable versus unstable linker may dependupon the toxicity of the cytotoxic and/or cytostatic agent. For agentsthat are toxic to normal cells, stable linkers are preferred. Agentsthat are selective or targeted and have lower toxicity to normal cellsmay utilize, chemical stability of the linker to the extracellularmilieu is less important. A wide variety of linkers useful for linkingdrugs to antibodies in the context of ADCs are known in the art. Any ofthese linkers, as well as other linkers, may be used to link thecytotoxic and/or cytostatic agents to the antibody of theanti-glyco-MUC1 ADCs of the disclosure.

Exemplary polyvalent linkers that may be used to link many cytotoxicand/or cytostatic agents to a single antibody molecule are described,for example, in WO 2009/073445; WO 2010/068795; WO 2010/138719; WO2011/120053; WO 2011/171020; WO 2013/096901; WO 2014/008375; WO2014/093379; WO 2014/093394; WO 2014/093640, the content of which areincorporated herein by reference in their entireties. For example, theFleximer linker technology developed by Mersana et al. has the potentialto enable high-DAR ADCs with good physicochemical properties. As shownbelow, the Mersana technology is based on incorporating drug moleculesinto a solubilizing poly-acetal backbone via a sequence of ester bonds.The methodology renders highly-loaded ADCs (DAR up to 20) whilemaintaining good physicochemical properties.

Additional examples of dendritic type linkers can be found in US2006/116422; US 2005/271615; de Groot et al. (2003) Angew. Chem. Int.Ed. 42:4490-4494; Amir et al. (2003) Angew. Chem. Int. Ed. 42:4494-4499;Shamis et al. (2004) J. Am. Chem. Soc. 126:1726-1731; Sun et al. (2002)Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003)Bioorganic & Medicinal Chemistry 11:1761-1768; King et al. (2002)Tetrahedron Letters 43:1987-1990, each of which is incorporated hereinby reference.

Exemplary monovalent linkers that may be used are described, forexample, in Nolting, 2013, Antibody-Drug Conjugates, Methods inMolecular Biology 1045:71-100; Kitson et al., 2013, CROs/CMOs—ChemicaOggi—Chemistry Today 31(4):30-38; Ducry et al., 2010, Bioconjugate Chem.21:5-13; Zhao et al., 2011, J. Med. Chem. 54:3606-3623; U.S. Pat. Nos.7,223,837; 8,568,728; 8,535,678; and WO2004010957, each of which isincorporated herein by reference.

By way of example and not limitation, some cleavable and noncleavablelinkers that may be included in the anti-glyco-MUC1 ADCs of thedisclosure are described below.

6.3.3. Cleavable Linkers

In certain embodiments, the linker selected is cleavable in vivo.Cleavable linkers may include chemically or enzymatically unstable ordegradable linkages. Cleavable linkers generally rely on processesinside the cell to liberate the drug, such as reduction in thecytoplasm, exposure to acidic conditions in the lysosome, or cleavage byspecific proteases or other enzymes within the cell. Cleavable linkersgenerally incorporate one or more chemical bonds that are eitherchemically or enzymatically cleavable while the remainder of the linkeris noncleavable. In certain embodiments, a linker comprises a chemicallylabile group such as hydrazone and/or disulfide groups. Linkerscomprising chemically labile groups exploit differential propertiesbetween the plasma and some cytoplasmic compartments. The intracellularconditions to facilitate drug release for hydrazone containing linkersare the acidic environment of endosomes and lysosomes, while thedisulfide containing linkers are reduced in the cytosol, which containshigh thiol concentrations, e.g., glutathione. In certain embodiments,the plasma stability of a linker comprising a chemically labile groupmay be increased by introducing steric hindrance using substituents nearthe chemically labile group.

Acid-labile groups, such as hydrazone, remain intact during systemiccirculation in the blood's neutral pH environment (pH 7.3-7.5) andundergo hydrolysis and release the drug once the ADC is internalizedinto mildly acidic endosomal (pH 5.0-6.5) and lysosomal (pH 4.5-5.0)compartments of the cell. This pH dependent release mechanism has beenassociated with nonspecific release of the drug. To increase thestability of the hydrazone group of the linker, the linker may be variedby chemical modification, e.g., substitution, allowing tuning to achievemore efficient release in the lysosome with a minimized loss incirculation.

Hydrazone-containing linkers may contain additional cleavage sites, suchas additional acid-labile cleavage sites and/or enzymatically labilecleavage sites. ADCs including exemplary hydrazone-containing linkersinclude the following structures:

wherein D and Ab represent the cytotoxic and/or cytostatic agent (drug)and Ab, respectively, and n represents the number of drug-linkers linkedto the antibody. In certain linkers such as linker (Ig), the linkercomprises two cleavable groups—a disulfide and a hydrazone moiety. Forsuch linkers, effective release of the unmodified free drug requiresacidic pH or disulfide reduction and acidic pH. Linkers such as (Ih) and(Ii) have been shown to be effective with a single hydrazone cleavagesite.

Additional linkers which remain intact during systemic circulation andundergo hydrolysis and release the drug when the ADC is internalizedinto acidic cellular compartments include carbonates. Such linkers canbe useful in cases where the cytotoxic and/or cytostatic agent can becovalently attached through an oxygen.

Other acid-labile groups that may be included in linkers includecis-aconityl-containing linkers. cis-Aconityl chemistry uses acarboxylic acid juxtaposed to an amide bond to accelerate amidehydrolysis under acidic conditions.

Cleavable linkers may also include a disulfide group. Disulfides arethermodynamically stable at physiological pH and are designed to releasethe drug upon internalization inside cells, wherein the cytosol providesa significantly more reducing environment compared to the extracellularenvironment. Scission of disulfide bonds generally requires the presenceof a cytoplasmic thiol cofactor, such as (reduced) glutathione (GSH),such that disulfide-containing linkers are reasonably stable incirculation, selectively releasing the drug in the cytosol. Theintracellular enzyme protein disulfide isomerase, or similar enzymescapable of cleaving disulfide bonds, may also contribute to thepreferential cleavage of disulfide bonds inside cells. GSH is reportedto be present in cells in the concentration range of 0.5-10 mM comparedwith a significantly lower concentration of GSH or cysteine, the mostabundant low-molecular weight thiol, in circulation at approximately 5Tumor cells, where irregular blood flow leads to a hypoxic state, resultin enhanced activity of reductive enzymes and therefore even higherglutathione concentrations. In certain embodiments, the in vivostability of a disulfide-containing linker may be enhanced by chemicalmodification of the linker, e.g., use of steric hindrance adjacent tothe disulfide bond.

ADCs including exemplary disulfide-containing linkers include thefollowing structures:

wherein D and Ab represent the drug and antibody, respectively, nrepresents the number of drug-linkers linked to the antibody and R isindependently selected at each occurrence from hydrogen or alkyl, forexample. In certain embodiments, increasing steric hindrance adjacent tothe disulfide bond increases the stability of the linker. Structuressuch as (ID and (II) show increased in vivo stability when one or more Rgroups is selected from a lower alkyl such as methyl.

Another type of cleavable linker that may be used is a linker that isspecifically cleaved by an enzyme. Such linkers are typicallypeptide-based or include peptidic regions that act as substrates forenzymes. Peptide based linkers tend to be more stable in plasma andextracellular milieu than chemically labile linkers. Peptide bondsgenerally have good serum stability, as lysosomal proteolytic enzymeshave very low activity in blood due to endogenous inhibitors and theunfavorably high pH value of blood compared to lysosomes. Release of adrug from an antibody occurs specifically due to the action of lysosomalproteases, e.g., cathepsin and plasmin. These proteases may be presentat elevated levels in certain tumor cells.

In exemplary embodiments, the cleavable peptide is selected fromtetrapeptides such as Gly-Phe-Leu-Gly (SEQ ID NO:128), Ala-Leu-Ala-Leu(SEQ ID NO:129) or dipeptides such as Val-Cit, Val-Ala, Met-(D)Lys,Asn-(D)Lys, Val-(D)Asp, Phe-Lys, Ile-Val, Asp-Val, His-Val,NorVal-(D)Asp, Ala-(D)Asp 5, Met-Lys, Asn-Lys, Ile-Pro, Me3Lys-Pro,PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys,Asn-(D)Lys, AM Met-(D)Lys, Asn-(D)Lys, AW Met-(D)Lys, and Asn-(D)Lys. Incertain embodiments, dipeptides are preferred over longer polypeptidesdue to hydrophobicity of the longer peptides.

A variety of dipeptide-based cleavable linkers useful for linking drugssuch as doxorubicin, mitomycin, camptothecin, pyrrolobenzodiazepine,tallysomycin and auristatin/auristatin family members to antibodies havebeen described (see, Dubowchik et al., 1998, J. Org. Chem. 67:1866-1872;Dubowchik et al., 1998, Bioorg. Med. Chem. Lett. 8(21):3341-3346; Walkeret al., 2002, Bioorg. Med. Chem. Lett. 12:217-219; Walker et al., 2004,Bioorg. Med. Chem. Lett. 14:4323-4327; Sutherland et al., 2013, Blood122: 1455-1463; and Francisco et al., 2003, Blood 102:1458-1465, of eachof which is incorporated herein by reference). All of these dipeptidelinkers, or modified versions of these dipeptide linkers, may be used inthe anti-glyco-MUC1 ADCs of the disclosure. Other dipeptide linkers thatmay be used include those found in ADCs such as Seattle Genetics'Brentuximab Vendotin SGN-35 (Adcetris™), Seattle Genetics SGN-75(anti-CD-70, Val-Cit-monomethyl auristatin F(MMAF), Seattle GeneticsSGN-CD33A (anti-CD-33, Val-Ala-(SGD-1882)), Celldex Therapeuticsglembatumumab (CDX-011) (anti-NMB, Val-Cit-monomethyl auristatin E(MMAE), and Cytogen PSMA-ADC (PSMA-ADC-1301) (anti-PSMA, Val-Cit-MMAE).

Enzymatically cleavable linkers may include a self-immolative spacer tospatially separate the drug from the site of enzymatic cleavage. Thedirect attachment of a drug to a peptide linker can result inproteolytic release of an amino acid adduct of the drug, therebyimpairing its activity. The use of a self-immolative spacer allows forthe elimination of the fully active, chemically unmodified drug uponamide bond hydrolysis.

One self-immolative spacer is the bifunctional para-aminobenzyl alcoholgroup, which is linked to the peptide through the amino group, formingan amide bond, while amine containing drugs may be attached throughcarbamate functionalities to the benzylic hydroxyl group of the linker(PABC). The resulting prodrugs are activated upon protease-mediatedcleavage, leading to a 1,6-elimination reaction releasing the unmodifieddrug, carbon dioxide, and remnants of the linker group. The followingscheme depicts the fragmentation of p-amidobenzyl ether and release ofthe drug:

wherein X-D represents the unmodified drug.

Heterocyclic variants of this self-immolative group have also beendescribed. See for example, U.S. Pat. No. 7,989,434, incorporated hereinby reference.

In some embodiments, the enzymatically cleavable linker is aβ-glucuronic acid-based linker. Facile release of the drug may berealized through cleavage of the β-glucuronide glycosidic bond by thelysosomal enzyme β-glucuronidase. This enzyme is present abundantlywithin lysosomes and is overexpressed in some tumor types, while theenzyme activity outside cells is low. β-Glucuronic acid-based linkersmay be used to circumvent the tendency of an ADC to undergo aggregationdue to the hydrophilic nature of β-glucuronides. In some embodiments,β-glucuronic acid-based linkers are preferred as linkers for ADCs linkedto hydrophobic drugs. The following scheme depicts the release of thedrug from and ADC containing a β-glucuronic acid-based linker:

A variety of cleavable β-glucuronic acid-based linkers useful forlinking drugs such as auristatins, camptothecin and doxorubicinanalogues, CBI minor-groove binders, and psymberin to antibodies havebeen described (see, see Nolting, Chapter 5 “Linker Technology inAntibody-Drug Conjugates,” In: Antibody-Drug Conjugates: Methods inMolecular Biology, vol. 1045, pp. 71-100, Laurent Ducry (Ed.), SpringerScience & Business Medica, LLC, 2013; Jeffrey et al., 2006, Bioconjug.Chem. 17:831-840; Jeffrey et al., 2007, Bioorg. Med. Chem. Lett.17:2278-2280; and Jiang et al., 2005, J. Am. Chem. Soc. 127:11254-11255,each of which is incorporated herein by reference). All of theseβ-glucuronic acid-based linkers may be used in the anti-glyco-MUC1 ADCsof the disclosure.

Additionally, cytotoxic and/or cytostatic agents containing a phenolgroup can be covalently bonded to a linker through the phenolic oxygen.One such linker, described in WO 2007/089149, relies on a methodology inwhich a diamino-ethane “SpaceLink” is used in conjunction withtraditional “PABO”-based self-immolative groups to deliver phenols. Thecleavage of the linker is depicted schematically below, where Drepresents a cytotoxic and/or cytostatic agent having a phenolichydroxyl group.

Cleavable linkers may include noncleavable portions or segments, and/orcleavable segments or portions may be included in an otherwisenon-cleavable linker to render it cleavable. By way of example only,polyethylene glycol (PEG) and related polymers may include cleavablegroups in the polymer backbone. For example, a polyethylene glycol orpolymer linker may include one or more cleavable groups such as adisulfide, a hydrazone or a dipeptide.

Other degradable linkages that may be included in linkers include esterlinkages formed by the reaction of PEG carboxylic acids or activated PEGcarboxylic acids with alcohol groups on a biologically active agent,wherein such ester groups generally hydrolyze under physiologicalconditions to release the biologically active agent. Hydrolyticallydegradable linkages include, but are not limited to, carbonate linkages;imine linkages resulting from reaction of an amine and an aldehyde;phosphate ester linkages formed by reacting an alcohol with a phosphategroup; acetal linkages that are the reaction product of an aldehyde andan alcohol; orthoester linkages that are the reaction product of aformate and an alcohol; and oligonucleotide linkages formed by aphosphoramidite group, including but not limited to, at the end of apolymer, and a 5′ hydroxyl group of an oligonucleotide.

In certain embodiments, the linker comprises an enzymatically cleavablepeptide moiety, for example, a linker comprising structural formula(IVa) or (IVb):

or a salt thereof, wherein: peptide represents a peptide (illustratedC→N and not showing the carboxy and amino “termini”) cleavable by alysosomal enzyme; T represents a polymer comprising one or more ethyleneglycol units or an alkylene chain, or combinations thereof; R^(a) isselected from hydrogen, alkyl, sulfonate and methyl sulfonate; p is aninteger ranging from 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1;

represents the point of attachment of the linker to a cytotoxic and/orcytostatic agent; and * represents the point of attachment to theremainder of the linker.

In certain embodiments, the peptide is selected from a tripeptide or adipeptide. In particular embodiments, the dipeptide is selected from:Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit;Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp;Ala-Val; Val-Ala; Phe-Lys; Val-Lys; Ala-Lys; Phe-Cit; Leu-Cit; Ile-Cit;Phe-Arg; and Trp-Cit. In certain embodiments, the dipeptide is selectedfrom: Cit-Val; and Ala-Val.

Specific exemplary embodiments of linkers according to structuralformula (IVa) that may be included in the anti-glyco-MUC1 ADCs of thedisclosure include the linkers illustrated below (as illustrated, thelinkers include a group suitable for covalently linking the linker to anantibody):

Specific exemplary embodiments of linkers according to structuralformula (IVb) that may be included in the anti-glyco-MUC1 ADCs of thedisclosure include the linkers illustrated below (as illustrated, thelinkers include a group suitable for covalently linking the linker to anantibody):

In certain embodiments, the linker comprises an enzymatically cleavablepeptide moiety, for example, a linker comprising structural formula(IVc) or (IVd):

or a salt thereof, wherein: peptide represents a peptide (illustratedC→N and not showing the carboxy and amino “termini”) cleavable by alysosomal enzyme; T represents a polymer comprising one or more ethyleneglycol units or an alkylene chain, or combinations thereof; R^(a) isselected from hydrogen, alkyl, sulfonate and methyl sulfonate; p is aninteger ranging from 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1; _x

represents the point of attachment of the linker to a cytotoxic and/orcytostatic agent; and * represents the point of attachment to theremainder of the linker.

Specific exemplary embodiments of linkers according to structuralformula (IVc) that may be included in the anti-glyco-MUC1 ADCs of thedisclosure include the linkers illustrated below (as illustrated, thelinkers include a group suitable for covalently linking the linker to anantibody):

Specific exemplary embodiments of linkers according to structuralformula (IVd) that may be included in the anti-glyco-MUC1 ADCs of thedisclosure include the linkers illustrated below (as illustrated, thelinkers include a group suitable for covalently linking the linker to anantibody):

In certain embodiments, the linker comprising structural formula (IVa),(IVb), (IVc), or (IVd) further comprises a carbonate moiety cleavable byexposure to an acidic medium. In particular embodiments, the linker isattached through an oxygen to a cytotoxic and/or cytostatic agent.

6.3.4. Non-Cleavable Linkers

Although cleavable linkers may provide certain advantages, the linkerscomprising the anti-glyco-MUC1 ADC of the disclosure need not becleavable. For noncleavable linkers, the release of drug does not dependon the differential properties between the plasma and some cytoplasmiccompartments. The release of the drug is postulated to occur afterinternalization of the ADC via antigen-mediated endocytosis and deliveryto lysosomal compartment, where the antibody is degraded to the level ofamino acids through intracellular proteolytic degradation. This processreleases a drug derivative, which is formed by the drug, the linker, andthe amino acid residue to which the linker was covalently attached. Theamino acid drug metabolites from conjugates with noncleavable linkersare more hydrophilic and generally less membrane permeable, which leadsto less bystander effects and less nonspecific toxicities compared toconjugates with a cleavable linker. In general, ADCs with noncleavablelinkers have greater stability in circulation than ADCs with cleavablelinkers. Non-cleavable linkers may be alkylene chains, or maybepolymeric in natures, such as, for example, based upon polyalkyleneglycol polymers, amide polymers, or may include segments of alkylenechains, polyalkylene glocols and/or amide polymers.

A variety of non-cleavable linkers used to link drugs to antibodies havebeen described. See, Jeffrey et al., 2006, Bioconjug. Chem. 17; 831-840;Jeffrey et al., 2007, Bioorg. Med. Chem. Lett. 17:2278-2280; and Jianget al., 2005, J. Am. Chem. Soc. 127:11254-11255, each of which isincorporated herein by reference. All of these linkers may be includedin the anti-glyco-MUC1 ADCs of the disclosure.

In certain embodiments, the linker is non-cleavable in vivo, for examplea linker according to structural formula (VIa), (VIb), (VIc) or (VId)(as illustrated, the linkers include a group suitable for covalentlylinking the linker to an antibody:

or salts thereof, wherein: R^(a) is selected from hydrogen, alkyl,sulfonate and methyl sulfonate; R^(x) is a moiety including a functionalgroup capable of covalently linking the linker to an antibody; and

represents the point of attachment of the linker to a cytotoxic and/orcytostatic agent.

Specific exemplary embodiments of linkers according to structuralformula (VIa)-(VId) that may be included in the anti-glyco-MUC1 ADCs ofthe disclosure include the linkers illustrated below (as illustrated,the linkers include a group suitable for covalently linking the linkerto an antibody, and

represents the point of attachment to a cytotoxic and/or cytostaticagent):

6.3.5. Groups Used to Attach Linkers to Antibodies

A variety of groups may be used to attach linker-drug synthons toantibodies to yield ADCs. Attachment groups can be electrophilic innature and include: maleimide groups, activated disulfides, activeesters such as NHS esters and HOBt esters, haloformates, acid halides,alkyl and benzyl halides such as haloacetamides. As discussed below,there are also emerging technologies related to “self-stabilizing”maleimides and “bridging disulfides” that can be used in accordance withthe disclosure. The specific group used will depend, in part, on thesite of attachment to the antibody.

One example of a “self-stabilizing” maleimide group that hydrolyzesspontaneously under antibody conjugation conditions to give an ADCspecies with improved stability is depicted in the schematic below. SeeUS20130309256 A1; also Lyon et al., Nature Biotech published online,doi:10.1038/nbt.2968.

Normal system:

Leads to “DAR loss” over time

SGN MalDPR (maleimido dipropylamino) system:

Polytherics has disclosed a method for bridging a pair of sulfhydrylgroups derived from reduction of a native hinge disulfide bond. See,Badescu et al., 2014, Bioconjugate Chem. 25:1124-1136. The reaction isdepicted in the schematic below. An advantage of this methodology is theability to synthesize enriched DAR4 ADCs by full reduction of IgGs (togive 4 pairs of sulfhydryls) followed by reaction with 4 equivalents ofthe alkylating agent. ADCs containing “bridged disulfides” are alsoclaimed to have increased stability.

Similarly, as depicted below, a maleimide derivative (1, below) that iscapable of bridging a pair of sulfhydryl groups has been developed. SeeWO2013/085925.

6.3.6. Linker Selection Considerations

As is known by skilled artisans, the linker selected for a particularADC may be influenced by a variety of factors, including but not limitedto, the site of attachment to the antibody (e.g., lys, cys or otheramino acid residues), structural constraints of the drug pharmacophoreand the lipophilicity of the drug. The specific linker selected for anADC should seek to balance these different factors for the specificantibody/drug combination. For a review of the factors that areinfluenced by choice of linkers in ADCs, see Nolting, Chapter 5 “LinkerTechnology in Antibody-Drug Conjugates,” In: Antibody-Drug Conjugates:Methods in Molecular Biology, vol. 1045, pp. 71-100, Laurent Ducry(Ed.), Springer Science & Business Medica, LLC, 2013.

For example, ADCs have been observed to effect killing of bystanderantigen-negative cells present in the vicinity of the antigen-positivetumor cells. The mechanism of bystander cell killing by ADCs hasindicated that metabolic products formed during intracellular processingof the ADCs may play a role. Neutral cytotoxic metabolites generated bymetabolism of the ADCs in antigen-positive cells appear to play a rolein bystander cell killing while charged metabolites may be preventedfrom diffusing across the membrane into the medium and therefore cannotaffect bystander killing. In certain embodiments, the linker is selectedto attenuate the bystander killing effect caused by cellular metabolitesof the ADC. In certain embodiments, the linker is selected to increasethe bystander killing effect.

The properties of the linker may also impact aggregation of the ADCunder conditions of use and/or storage. Typically, ADCs reported in theliterature contain no more than 3-4 drug molecules per antibody molecule(see, e.g., Chari, 2008, Acc Chem Res 41:98-107). Attempts to obtainhigher drug-to-antibody ratios (“DAR”) often failed, particularly ifboth the drug and the linker were hydrophobic, due to aggregation of theADC (King et al., 2002, J Med Chem 45:4336-4343; Hollander et al., 2008,Bioconjugate Chem 19:358-361; Burke et al., 2009 Bioconjugate Chem20:1242-1250). In many instances, DARs higher than 3-4 could bebeneficial as a means of increasing potency. In instances where thecytotoxic and/or cytostatic agent is hydrophobic in nature, it may bedesirable to select linkers that are relatively hydrophilic as a meansof reducing ADC aggregation, especially in instances where DARS greaterthan 3-4 are desired. Thus, in certain embodiments, the linkerincorporates chemical moieties that reduce aggregation of the ADCsduring storage and/or use. A linker may incorporate polar or hydrophilicgroups such as charged groups or groups that become charged underphysiological pH to reduce the aggregation of the ADCs. For example, alinker may incorporate charged groups such as salts or groups thatdeprotonate, e.g., carboxylates, or protonate, e.g., amines, atphysiological pH.

Exemplary polyvalent linkers that have been reported to yield DARs ashigh as 20 that may be used to link numerous cytotoxic and/or cytostaticagents to an antibody are described in WO 2009/073445; WO 2010/068795;WO 2010/138719; WO 2011/120053; WO 2011/171020; WO 2013/096901; WO2014/008375; WO 2014/093379; WO 2014/093394; WO 2014/093640, the contentof which are incorporated herein by reference in their entireties.

In particular embodiments, the aggregation of the ADCs during storage oruse is less than about 10% as determined by size-exclusionchromatography (SEC). In particular embodiments, the aggregation of theADCs during storage or use is less than 10%, such as less than about 5%,less than about 4%, less than about 3%, less than about 2%, less thanabout 1%, less than about 0.5%, less than about 0.1%, or even lower, asdetermined by size-exclusion chromatography (SEC).

6.3.7. Methods of Making Anti-Glyco-MUC1 ADCs

The anti-glyco-MUC1 ADCs of the disclosure may be synthesized usingchemistries that are well-known. The chemistries selected will dependupon, among other things, the identity of the cytotoxic and/orcytostatic agent(s), the linker and the groups used to attach linker tothe antibody. Generally, ADCs according to formula (I) may be preparedaccording to the following scheme:

D-L-R^(x)+Ab-R^(y)→[D-L-XY]_(n)-Ab   (I)

where D, L, Ab, XY and n are as previously defined, and R^(x) and R^(y)represent complementary groups capable of forming a covalent linkageswith one another, as discussed above.

The identities of groups R^(x) and R^(y) will depend upon the chemistryused to link synthon D-L-R^(x) to the antibody. Generally, the chemistryused should not alter the integrity of the antibody, for example itsability to bind its target. Preferably, the binding properties of theconjugated antibody will closely resemble those of the unconjugatedantibody. A variety of chemistries and techniques for conjugatingmolecules to biological molecules such as antibodies are known in theart and in particular to antibodies, are well-known. See, e.g., Amon etal., “Monoclonal Antibodies For Immunotargeting Of Drugs In CancerTherapy,” in: Monoclonal Antibodies And Cancer Therapy, Reisfeld et al.Eds., Alan R. Liss, Inc., 1985; Hellstrom et al., “Antibodies For DrugDelivery,” in: Controlled Drug Delivery, Robinson et al. Eds., MarcelDekker, Inc., 2nd Ed. 1987; Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review,” in: Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al., Eds., 1985;“Analysis, Results, and Future Prospective of the Therapeutic Use ofRadiolabeled Antibody In Cancer Therapy,” in: Monoclonal Antibodies ForCancer Detection And Therapy, Baldwin et al., Eds., Academic Press,1985; Thorpe et al., 1982, Immunol. Rev. 62:119-58; PCT publication WO89/12624. Any of these chemistries may be used to link the synthons toan antibody.

A number of functional groups R^(x) and chemistries useful for linkingsynthons to accessible lysine residues are known, and include by way ofexample and not limitation NHS-esters and isothiocyanates.

A number of functional groups R^(x) and chemistries useful for linkingsynthons to accessible free sulfhydryl groups of cysteine residues areknown, and include by way of example and not limitation haloacetyls andmaleimides.

However, conjugation chemistries are not limited to available side chaingroups. Side chains such as amines may be converted to other usefulgroups, such as hydroxyls, by linking an appropriate small molecule tothe amine. This strategy can be used to increase the number of availablelinking sites on the antibody by conjugating multifunctional smallmolecules to side chains of accessible amino acid residues of theantibody. Functional groups R^(x) suitable for covalently linking thesynthons to these “converted” functional groups are then included in thesynthons.

The antibody may also be engineered to include amino acid residues forconjugation. An approach for engineering antibodies to includenon-genetically encoded amino acid residues useful for conjugating drugsin the context of ADCs is described by Axup et al., 2012, Proc Natl AcadSci USA. 109(40):16101-16106, as are chemistries and functional groupuseful for linking synthons to the non-encoded amino acids.

Typically, the synthons are linked to the side chains of amino acidresidues of the antibody, including, for example, the primary aminogroup of accessible lysine residues or the sulfhydryl group ofaccessible cysteine residues. Free sulfhydryl groups may be obtained byreducing interchain disulfide bonds.

For linkages where R^(y) is a sulfhydryl group (for example, when R^(x)is a maleimide), the antibody is generally first fully or partiallyreduced to disrupt interchain disulfide bridges between cysteineresidues.

Cysteine residues that do not participate in disulfide bridges mayengineered into an antibody by mutation of one or more codons. Reducingthese unpaired cysteines yields a sulfhydryl group suitable forconjugation. Preferred positions for incorporating engineered cysteinesinclude, by way of example and not limitation, positions S112C, S113C,A114C, S115C, A176C, 5180C, S252C, V286C, V292C, S357C, A359C, S398C,S428C (Kabat numbering) on the human IgG₁ heavy chain and positionsV110C, S114C, S121C, S127C, S168C, V205C (Kabat numbering) on the humanIg kappa light chain (see, e.g., U.S. Pat. Nos. 7,521,541, 7,855,275 and8,455,622).

As will appreciated by skilled artisans, the number of cytotoxic and/orcytostatic agents linked to an antibody molecule may vary, such that acollection of ADCs may be heterogeneous in nature, where some antibodiescontain one linked agent, some two, some three, etc. (and some none).The degree of heterogeneity will depend upon, among other things, thechemistries used for linking the cytotoxic and/or cytostatic agents. Forexample, where the antibodies are reduced to yield sulfhydryl groups forattachment, heterogeneous mixtures of antibodies having zero, 2, 4, 6 or8 linked agents per molecule are often produced. Furthermore, bylimiting the molar ratio of attachment compound, antibodies having zero,1, 2, 3, 4, 5, 6, 7 or 8 linked agents per molecule are often produced.Thus, it will be understood that depending upon context, stated DARs maybe averages for a collection of antibodies. For example, “DAR4” canrefer to an ADC preparation that has not been subjected to purificationto isolate specific DAR peaks and can comprise a heterogeneous mixtureof ADC molecules having different numbers of cytostatic and/or cytotoxicagents attached per antibody (e.g., 0, 2, 4, 6, 8 agents per antibody),but has an average drug-to-antibody ratio of 4. Similarly, in someembodiments, “DAR2” refers to a heterogeneous ADC preparation in whichthe average drug-to-antibody ratio is 2.

When enriched preparations are desired, antibodies having definednumbers of linked cytotoxic and/or cytostatic agents may be obtained viapurification of heterogeneous mixtures, for example, via columnchromatography, e.g., hydrophobic interaction chromatography.

Purity may be assessed by a variety of methods, as is known in the art.As a specific example, an ADC preparation may be analyzed via HPLC orother chromatography and the purity assessed by analyzing areas underthe curves of the resultant peaks.

6.4 Chimeric Antigen Receptors

The present disclosure provides chimeric antigen receptors (CARs)comprising the anti-glyco-MUC1 antibodies or antigen-binding fragmentsdescribed herein.

The CARs of the disclosure typically comprise an extracellular domainoperably linked to a transmembrane domain which is in turn operablylinked to an intracellular domain for signaling.

The extracellular domains of the CARs of the disclosure comprise thesequence of an anti-glyco-MUC1 antibody or antigen-binding fragment(e.g., as described in Section 6.1 or embodiments 1 to 90).

Exemplary transmembrane domain sequence and intracellular domainsequences are described in Section 6.4.1 and 6.4.2, respectively.

Several fusion proteins described herein (e.g., embodiments 92 and94-96) are CARs, and the CAR-related disclosures apply to such fusionproteins.

6.4.1. Transmembrane Domain

With respect to the transmembrane domain, the CAR can be designed tocomprise a transmembrane domain that is operably linked (e.g., fused) tothe extracellular domain of the CAR.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsof particular use in this disclosure may be derived from (i.e., compriseat least the transmembrane region(s) of) the alpha, beta or zeta chainof the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In someinstances, a variety of human hinges can be employed as well includingthe human Ig (immunoglobulin) hinge.

In one embodiment, the transmembrane domain is synthetic (i.e.,non-naturally occurring). Examples of synthetic transmembrane domainsare peptides comprising predominantly hydrophobic residues such asleucine and valine. Preferably a triplet of phenylalanine, tryptophanand valine will be found at each end of a synthetic transmembranedomain. Optionally, a short oligo- or polypeptide linker, preferablybetween 2 and 10 amino acids in length may form the linkage between thetransmembrane domain and the cytoplasmic signaling domain of the CAR. Aglycine-serine doublet provides a particularly suitable linker.

In one embodiment, the transmembrane domain in the CAR of the disclosureis the CD8 transmembrane domain. In one embodiment, the CD8transmembrane domain comprises the amino acid sequenceYLHLGALGRDLWGPSPVTGYHPLL.

In one embodiment, the transmembrane domain in the CAR of the disclosureis the CD28 transmembrane domain. In one embodiment, the CD28transmembrane domain comprises the amino acid sequenceFWVLVVVGGVLACYSLLVTVAFIIFWV.

In some instances, the transmembrane domain of the CAR of the disclosurecomprises the CD8a hinge domain. In one embodiment, the CD8a hingedomain comprises the amino acid sequenceTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC.

6.4.2. Intracellular Domain

The intracellular signaling domain of the CAR of the disclosure isresponsible for activation of at least one of the normal effectorfunctions of the immune cell in which the CAR is expressed. The term“effector function” refers to a specialized function of a cell. Effectorfunction of a T cell, for example, may be cytolytic activity or helperactivity including the secretion of cytokines. Thus the term“intracellular signaling domain” refers to the portion of a proteinwhich transduces the effector function signal and directs the cell toperform a specialized function. While usually the entire intracellularsignaling domain can be employed, in many cases it is not necessary touse the entire chain. To the extent that a truncated portion of theintracellular signaling domain is used, such truncated portion may beused in place of the intact chain as long as it transduces the effectorfunction signal. The term intracellular signaling domain is thus meantto include any truncated portion of the intracellular signaling domainsufficient to transduce the effector function signal.

Preferred examples of intracellular signaling domains for use in the CARof the disclosure include the cytoplasmic sequences of the T cellreceptor (TCR) and co-receptors that act in concert to initiate signaltransduction following antigen receptor engagement, as well as anyderivative or variant of these sequences and any synthetic sequence thathas the same functional capability.

Signals generated through the TCR alone may be insufficient for fullactivation of the T cell and a secondary or co-stimulatory signal isalso required. Thus, T cell activation can be said to be mediated by twodistinct classes of cytoplasmic signaling sequence: those that initiateantigen-dependent primary activation through the TCR (primarycytoplasmic signaling sequences) and those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal (secondary cytoplasmic signaling sequences).

Primary cytoplasmic signaling sequences regulate primary activation ofthe TCR complex either in a stimulatory way, or in an inhibitory way.Primary cytoplasmic signaling sequences that act in a stimulatory mannermay contain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary cytoplasmic signaling sequences thatare of particular use in the CARs of the disclosure include thosederived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d. It is particularlypreferred that cytoplasmic signaling molecule in the CAR of thedisclosure comprises a cytoplasmic signaling sequence from CD3-zeta.

In a preferred embodiment, the cytoplasmic domain of the CAR is designedto include an ITAM containing primary cytoplasmic signaling sequencesdomain (e.g., that of CD3-zeta) by itself or combined with any otherdesired cytoplasmic domain(s) useful in the context of the CAR of thedisclosure. For example, the cytoplasmic domain of the CAR can include aCD3 zeta chain portion and a costimulatory signaling region.

The costimulatory signaling region refers to a portion of the CARcomprising the intracellular domain of a costimulatory molecule. Acostimulatory molecule is a cell surface molecule other than an antigenreceptor or its ligands that is required for an efficient response oflymphocytes to an antigen. Examples of such molecules include CD27,CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and a ligand that specifically binds with CD83, and the like.

The cytoplasmic signaling sequences within the cytoplasmic signalingportion of the CAR of the disclosure may be linked to each other in arandom or specified order. Optionally, a short oligo- or polypeptidelinker, preferably between 2 and 10 amino acids in length may form thelinkage. A glycine-serine doublet provides a particularly suitablelinker.

In one embodiment, the cytoplasmic domain comprises the signaling domainof CD3-zeta and the signaling domain of CD28. In another embodiment, thecytoplasmic domain comprises the signaling domain of CD3-zeta and thesignaling domain of 4-1BB.

6.5 Nucleic Acids, Recombinant Vectors and Host Cells

The present disclosure encompasses nucleic acid molecules encodingimmunoglobulin light and heavy chain genes for anti-glyco-MUC1antibodies, vectors comprising such nucleic acids, and host cellscapable of producing the anti-glyco-MUC1 antibodies of the disclosure.In certain aspects, the nucleic acid molecules encode, and the hostcells are capable of expressing, the anti-glyco-MUC1 antibodies andantibody-binding fragments of the disclosure (e.g., as described inSection 6.1 and embodiments 1 to 90) as well as fusion proteins (e.g.,as described in embodiments 91-96) and chimeric antigen receptors (e.g.,as described in Section 6.4 and embodiments 97-98) containing them.Exemplary vectors of the disclosure are described in embodiments 111-113and exemplary host cells are described in embodiments 114-117.

An anti-glyco-MUC1 antibody of the disclosure can be prepared byrecombinant expression of immunoglobulin light and heavy chain genes ina host cell. To express an antibody recombinantly, a host cell istransfected with one or more recombinant expression vectors carrying DNAfragments encoding the immunoglobulin light and heavy chains of theantibody such that the light and heavy chains are expressed in the hostcell and, optionally, secreted into the medium in which the host cellsare cultured, from which medium the antibodies can be recovered.Standard recombinant DNA methodologies are used to obtain antibody heavyand light chain genes, incorporate these genes into recombinantexpression vectors and introduce the vectors into host cells, such asthose described in Molecular Cloning; A Laboratory Manual, SecondEdition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, N.Y., 1989), Current Protocols in Molecular Biology (Ausubel, F. M. etal., eds., Greene Publishing Associates, 1989) and in U.S. Pat. No.4,816,397.

To generate nucleic acids encoding such anti-glyco-MUC1 antibodies, DNAfragments encoding the light and heavy chain variable regions are firstobtained. These DNAs can be obtained by amplification and modificationof germline DNA or cDNA encoding light and heavy chain variablesequences, for example using the polymerase chain reaction (PCR).Germline DNA sequences for human heavy and light chain variable regiongenes are known in the art (See, e.g., the “VBASE” human germlinesequence database; see also Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson et al., 1992, J.Mol. Biol. 22T:116-198; and Cox et al., 1994, Eur. J. Immunol.24:827-836; the contents of each of which are incorporated herein byreference).

Once DNA fragments encoding anti-glyco-MUC1 antibody-related V_(H) andV_(L) segments are obtained, these DNA fragments can be furthermanipulated by standard recombinant DNA techniques, for example toconvert the variable region genes to full-length antibody chain genes,to Fab fragment genes or to a scFv gene. In these manipulations, aV_(H)- or V_(L)-encoding DNA fragment is operatively linked to anotherDNA fragment encoding another protein, such as an antibody constantregion or a flexible linker. The term “operatively linked,” as used inthis context, is intended to mean that the two DNA fragments are joinedsuch that the amino acid sequences encoded by the two DNA fragmentsremain in-frame.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH₁,CH₂, CH₃ and, optionally, CH₄). The sequences of human heavy chainconstant region genes are known in the art (See, e.g., Kabat et al.,1991, Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242) and DNA fragments encompassing these regions can be obtained bystandard PCR amplification. The heavy chain constant region can be anIgG₁, IgG₂, IgG₃, IgG₄, IgA, IgE, IgM or IgD constant region, but incertain embodiments is an IgG₁ or IgG₄ constant region. For a Fabfragment heavy chain gene, the V_(H)-encoding DNA can be operativelylinked to another DNA molecule encoding only the heavy chain CH1constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CL. The sequences of humanlight chain constant region genes are known in the art (See, e.g., Kabatet al., 1991, Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region, but in certain embodiments isa kappa constant region.

To create a scFv gene, the V_(H)- and V_(L)-encoding DNA fragments canbe operatively linked to another fragment encoding a flexible linker,e.g., encoding the amino acid sequence (Gly₄˜Ser)₃, such that the V_(H)and V_(L) sequences can be expressed as a contiguous single-chainprotein, with the V_(H) and V_(L) regions joined by the flexible linker(See, e.g., Bird et al., 1988, Science 242:423-426; Huston et al., 1988,Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature348:552-554).

To express the anti-glyco-MUC1 antibodies of the disclosure, DNAsencoding partial or full-length light and heavy chains, obtained asdescribed above, are inserted into expression vectors such that thegenes are operatively linked to transcriptional and translationalcontrol sequences. In this context, the term “operatively linked” isintended to mean that an antibody gene is ligated into a vector suchthat transcriptional and translational control sequences within thevector serve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vectors or, more typically, bothgenes are inserted into the same expression vector.

The antibody genes are inserted into the expression vector by standardmethods (e.g., ligation of complementary restriction sites on theantibody gene fragment and vector, or blunt end ligation if norestriction sites are present). Prior to insertion of theanti-glyco-MUC1 antibody-related light or heavy chain sequences, theexpression vector can already carry antibody constant region sequences.For example, one approach to converting the anti-glyco-MUC1 monoclonalantibody-related V_(H) and V_(L) sequences to full-length antibody genesis to insert them into expression vectors already encoding heavy chainconstant and light chain constant regions, respectively, such that theV_(H) segment is operatively linked to the CH segment(s) within thevector and the V_(L) segment is operatively linked to the CL segmentwithin the vector. Additionally or alternatively, the recombinantexpression vector can encode a signal peptide that facilitates secretionof the antibody chain from a host cell. The antibody chain gene can becloned into the vector such that the signal peptide is linked in-frameto the amino terminus of the antibody chain gene. The signal peptide canbe an immunoglobulin signal peptide or a heterologous signal peptide(i.e., a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the disclosure carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif., 1990. It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Suitable regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, see,e.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al., and U.S. Pat. No. 4,968,615 by Schaffner et al.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the disclosure can carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (See, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Suitable selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in DHFR⁻ host cells withmethotrexate selection/amplification) and the neo gene (for G418selection). For expression of the light and heavy chains, the expressionvector(s) encoding the heavy and light chains is transfected into a hostcell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, lipofection,calcium-phosphate precipitation, DEAE—dextran transfection and the like.

It is possible to express the antibodies of the disclosure in eitherprokaryotic or eukaryotic host cells. In certain embodiments, expressionof antibodies is performed in eukaryotic cells, e.g., mammalian hostcells, of optimal secretion of a properly folded and immunologicallyactive antibody. Exemplary mammalian host cells for expressing therecombinant antibodies of the disclosure include Chinese Hamster Ovary(CHO cells) (including DHFR⁻ CHO cells, described in Urlaub and Chasin,1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFRselectable marker, e.g., as described in Kaufman and Sharp, 1982, Mol.Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can berecovered from the culture medium using standard protein purificationmethods. Host cells can also be used to produce portions of intactantibodies, such as Fab fragments or scFv molecules. It is understoodthat variations on the above procedure are within the scope of thepresent disclosure. For example, it can be desirable to transfect a hostcell with DNA encoding either the light chain or the heavy chain (butnot both) of an anti-glyco-MUC1 antibody of this disclosure.

For expression of a CAR of the disclosure, for example as described inSection 6.4 and in embodiments 97 and 98, it is preferably that the hostcell is a T cell, preferably a human T cell. In some embodiments, thehost cell exhibits an anti-tumor immunity when the cell is cross-linkedwith MUC1 on a tumor cell. Detailed methods for producing the T cells ofthe disclosure are described in Section 6.5.1

Recombinant DNA technology can also be used to remove some or all of theDNA encoding either or both of the light and heavy chains that is notnecessary for binding to glyco-MUC1. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of thedisclosure.

For recombinant expression of an anti-glyco-MUC1 antibody of thedisclosure, the host cell can be co-transfected with two expressionvectors of the disclosure, the first vector encoding a heavy chainderived polypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors can contain identical selectable markers,or they can each contain a separate selectable marker. Alternatively, asingle vector can be used which encodes both heavy and light chainpolypeptides.

Once a nucleic acid encoding one or more portions of an anti-glyco-MUC1antibody, further alterations or mutations can be introduced into thecoding sequence, for example to generate nucleic acids encodingantibodies with different CDR sequences, antibodies with reducedaffinity to the Fc receptor, or antibodies of different subclasses.

The anti-glyco-MUC1 antibodies of the disclosure can also be produced bychemical synthesis (e.g., by the methods described in Solid PhasePeptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford,Ill.). Variant antibodies can also be generated using a cell-freeplatform (See, e.g., Chu et al., Biochemia No. 2, 2001 (Roche MolecularBiologicals) and Murray et al., 2013, Current Opinion in ChemicalBiology, 17:420-426).

Once an anti-glyco-MUC1 antibody of the disclosure has been produced byrecombinant expression, it can be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theanti-glyco-MUC1 antibodies of the present disclosure and/or bindingfragments can be fused to heterologous polypeptide sequences describedherein or otherwise known in the art to facilitate purification.

Once isolated, the anti-glyco-MUC1 antibody can, if desired, be furtherpurified, e.g., by high performance liquid chromatography (see, e.g.,Fisher, Laboratory Techniques In Biochemistry And Molecular Biology,Work and Burdon, eds., Elsevier, 1980), or by gel filtrationchromatography on a Superdex™ 75 column (Pharmacia Biotech AB, Uppsala,Sweden).

6.5.1. Recombinant Production of CARs in T Cells

In some embodiments, nucleic acids encoding the anti-glyco-MUC1 CARs ofthe disclosure are delivered into cells using a retroviral or lentiviralvector. CAR-expressing retroviral and lentiviral vectors can bedelivered into different types of eukaryotic cells as well as intotissues and whole organisms using transduced cells as carriers orcell-free local or systemic delivery of encapsulated, bound or nakedvectors. The method used can be for any purpose where stable expressionis required or sufficient.

In other embodiments, the CAR sequences are delivered into cells usingin vitro transcribed mRNA. In vitro transcribed mRNA CAR can bedelivered into different types of eukaryotic cells as well as intotissues and whole organisms using transfected cells as carriers orcell-free local or systemic delivery of encapsulated, bound or nakedmRNA. The method used can be for any purpose where transient expressionis required or sufficient.

In another embodiment, the desired CAR can be expressed in the cells byway of transponsons.

One advantage of RNA transfection methods of the disclosure is that RNAtransfection is essentially transient and a vector-free: an RNAtransgene can be delivered to a lymphocyte and expressed thereinfollowing a brief in vitro cell activation, as a minimal expressingcassette without the need for any additional viral sequences. Underthese conditions, integration of the transgene into the host cell genomeis unlikely. Cloning of cells is not necessary because of the efficiencyof transfection of the RNA and its ability to uniformly modify theentire lymphocyte population.

Genetic modification of T cells with in vitro-transcribed RNA (IVT-RNA)makes use of two different strategies both of which have beensuccessively tested in various animal models. Cells are transfected within vitro-transcribed RNA by means of lipofection or electroporation.Preferably, it is desirable to stabilize IVT-RNA using variousmodifications in order to achieve prolonged expression of transferredIVT-RNA.

Some IVT vectors are known in the literature which are utilized in astandardized manner as template for in vitro transcription and whichhave been genetically modified in such a way that stabilized RNAtranscripts are produced. Currently protocols used in the art are basedon a plasmid vector with the following structure: a 5′ RNA polymerasepromoter enabling RNA transcription, followed by a gene of interestwhich is flanked either 3′ and/or 5′ by untranslated regions (UTR), anda 3′ polyadenyl cassette containing 50-70 A nucleotides. Prior to invitro transcription, the circular plasmid is linearized downstream ofthe polyadenyl cassette by type II restriction enzymes (recognitionsequence corresponds to cleavage site). The polyadenyl cassette thuscorresponds to the later poly(A) sequence in the transcript. As a resultof this procedure, some nucleotides remain as part of the enzymecleavage site after linearization and extend or mask the poly (A)sequence at the 3′ end. It is not clear, whether this nonphysiologicaloverhang affects the amount of protein produced intracellularly fromsuch a construct.

RNA has several advantages over more traditional plasmid or viralapproaches. Gene expression from an RNA source does not requiretranscription and the protein product is produced rapidly after thetransfection. Further, since the RNA has to only gain access to thecytoplasm, rather than the nucleus, and therefore typical transfectionmethods result in an extremely high rate of transfection. In addition,plasmid based approaches require that the promoter driving theexpression of the gene of interest be active in the cells under study.

In another aspect, the RNA construct can be delivered into the cells byelectroporation. See, e.g., the formulations and methodology ofelectroporation of nucleic acid constructs into mammalian cells astaught in US 2004/0014645, US 2005/0052630A1, US 2005/0070841A1, US2004/0059285A1, US 2004/0092907A1. The various parameters includingelectric field strength required for electroporation of any known celltype are generally known in the relevant research literature as well asnumerous patents and applications in the field. See e.g., U.S. Pat. Nos.6,678,556, 7,171,264, and 7,173,116. Apparatus for therapeuticapplication of electroporation are available commercially, e.g., theMedPulser™ DNA Electroporation Therapy System (Inovio/Genetronics, SanDiego, Calif.), and are described in patents such as U.S. Pat. Nos.6,567,694; 6,516,223, 5,993,434, 6,181,964, 6,241,701, and 6,233,482;electroporation may also be used for transfection of cells in vitro asdescribed e.g. in US20070128708A1. Electroporation may also be utilizedto deliver nucleic acids into cells in vitro. Accordingly,electroporation-mediated administration into cells of nucleic acidsincluding expression constructs utilizing any of the many availabledevices and electroporation systems known to those of skill in the artpresents an exciting new means for delivering an RNA of interest to atarget cell.

6.5.1.1 Sources of T Cells

Prior to expansion and genetic modification, a source of T cells isobtained from a subject. The term “subject” is intended to includeliving organisms in which an immune response can be elicited (e.g.,mammals). Examples of subjects include humans, dogs, cats, mice, rats,and transgenic species thereof. Preferably, subjects are human.

T cells can be obtained from a number of sources, including peripheralblood mononuclear cells, bone marrow, lymph node tissue, cord blood,thymus tissue, tissue from a site of infection, ascites, pleuraleffusion, spleen tissue, and tumors. In certain embodiments of thepresent disclosure, any number of T cell lines available in the art, maybe used. In certain embodiments of the present disclosure, T cells canbe obtained from a unit of blood collected from a subject using anynumber of techniques known to the skilled artisan, such as Ficoll™separation. In one preferred embodiment, cells from the circulatingblood of an individual are obtained by apheresis. The apheresis producttypically contains lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and platelets. In one embodiment, the cells collected byapheresis may be washed to remove the plasma fraction and to place thecells in an appropriate buffer or media for subsequent processing steps.In one embodiment of the disclosure, the cells are washed with phosphatebuffered saline (PBS). In an alternative embodiment, the wash solutionlacks calcium and may lack magnesium or may lack many if not alldivalent cations. Again, surprisingly, initial activation steps in theabsence of calcium lead to magnified activation. As those of ordinaryskill in the art would readily appreciate a washing step may beaccomplished by methods known to those in the art, such as by using asemi-automated “flow-through” centrifuge (for example, the Cobe 2991cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5)according to the manufacturers instructions. After washing, the cellsmay be resuspended in a variety of biocompatible buffers, such as, forexample, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solutionwith or without buffer. Alternatively, the undesirable components of theapheresis sample may be removed and the cells directly resuspended inculture media.

In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient or bycounterflow centrifugal elutriation. A specific subpopulation of Tcells, such as CD3⁺, CD28′, CD4⁺, CD8⁺, CD45RA⁺ and CD45RO⁺ T cells, canbe further isolated by positive or negative selection techniques. Forexample, in one embodiment, T cells are isolated by incubation withanti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, such as DYNABEADS®M-450 CD3/CD28 T, for a time period sufficient for positive selection ofthe desired T cells. In one embodiment, the time period is about 30minutes. In a further embodiment, the time period ranges from 30 minutesto 36 hours or longer and all integer values there between. In a furtherembodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. Inyet another preferred embodiment, the time period is 10 to 24 hours. Inone preferred embodiment, the incubation time period is 24 hours. Forisolation of T cells from patients with leukemia, use of longerincubation times, such as 24 hours, can increase cell yield. Longerincubation times may be used to isolate T cells in any situation wherethere are few T cells as compared to other cell types, such in isolatingtumor infiltrating lymphocytes (TIL) from tumor tissue or fromimmunocompromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or, lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this disclosure. In certainembodiments, it may be desirable to perform the selection procedure anduse the “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4⁺ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD11 b, CD16,HLA-DR, and CD8. In certain embodiments, it may be desirable to enrichfor or positively select for regulatory T cells which typically expressCD4⁺, CD25⁺, CD62L^(hi), GITR⁺, and FoxP3⁺. Alternatively, in certainembodiments, T regulatory cells are depleted by anti-C25 conjugatedbeads or other similar method of selection.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (i.e., leukemic blood, tumortissue, etc.). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8⁺ T cells that normally haveweaker CD28 expression.

In a related embodiment, it may be desirable to use lower concentrationsof cells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4⁺ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8⁺ T cells in dilute concentrations. In one embodiment, theconcentration of cells used is 5×10⁶/ml. In other embodiments, theconcentration used can be from about 1×10⁵/ml to 1×10⁶/ml, and anyinteger value in between.

In other embodiments, the cells may be incubated on a rotator forvarying lengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen.

In certain embodiments, cryopreserved cells are thawed and washed asdescribed herein and allowed to rest for one hour at room temperatureprior to activation using the methods of the present disclosure.

Also contemplated in the context of the disclosure is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T cells, isolated and frozenfor later use in T cell therapy for any number of diseases or conditionsthat would benefit from T cell therapy, such as those described herein.In one embodiment a blood sample or an apheresis is taken from agenerally healthy subject. In certain embodiments, a blood sample or anapheresis is taken from a generally healthy subject who is at risk ofdeveloping a disease, but who has not yet developed a disease, and thecells of interest are isolated and frozen for later use. In certainembodiments, the T cells may be expanded, frozen, and used at a latertime. In certain embodiments, samples are collected from a patientshortly after diagnosis of a particular disease as described herein butprior to any treatments. In a further embodiment, the cells are isolatedfrom a blood sample or an apheresis from a subject prior to any numberof relevant treatment modalities, including but not limited to treatmentwith agents such as natalizumab, efalizumab, antiviral agents,chemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies,cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid,steroids, FR901228, and irradiation. These drugs inhibit either thecalcium dependent phosphatase calcineurin (cyclosporine and FK506) orinhibit the p70S6 kinase that is important for growth factor inducedsignaling (rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson etal., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun.5:763-773, 1993). In a further embodiment, the cells are isolated for apatient and frozen for later use in conjunction with (e.g., before,simultaneously or following) bone marrow or stem cell transplantation orT cell ablative therapy using either chemotherapy agents such as,fludarabine, external-beam radiation therapy (XRT), cyclophosphamide.

In a further embodiment of the present disclosure, T cells are obtainedfrom a patient directly following treatment. In this regard, it has beenobserved that following certain cancer treatments, in particulartreatments with drugs that damage the immune system, shortly aftertreatment during the period when patients would normally be recoveringfrom the treatment, the quality of T cells obtained may be optimal orimproved for their ability to expand ex vivo. Likewise, following exvivo manipulation using the methods described herein, these cells may bein a preferred state for enhanced engraftment and in vivo expansion.Thus, it is contemplated within the context of the present disclosure tocollect blood cells, including T cells, dendritic cells, or other cellsof the hematopoietic lineage, during this recovery phase. Further, incertain embodiments, mobilization (for example, mobilization withGM-CSF) and conditioning regimens can be used to create a condition in asubject wherein repopulation, recirculation, regeneration, and/orexpansion of particular cell types is favored, especially during adefined window of time following therapy. Illustrative cell typesinclude T cells, B cells, dendritic cells, and other cells of the immunesystem.

6.5.1.2 Activation and Expansion of T Cells

T cells are activated and expanded generally using methods as described,for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680;6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318;7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514;6,867,041; and U.S. Patent Application Publication No. 20060121005.

Generally, the T cells of the disclosure are expanded by contact with asurface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a co-stimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4⁺ T cells or CD8⁺ T cells, an anti-CD3 antibody and ananti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besancon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328,1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In certain embodiments, the primary stimulatory signal and theco-stimulatory signal for the T cell may be provided by differentprotocols. For example, the agents providing each signal may be insolution or coupled to a surface. When coupled to a surface, the agentsmay be coupled to the same surface (i.e., in “cis” formation) or toseparate surfaces (i.e., in “trans” formation). Alternatively, one agentmay be coupled to a surface and the other agent in solution. In oneembodiment, the agent providing the co-stimulatory signal is bound to acell surface and the agent providing the primary activation signal is insolution or coupled to a surface. In certain embodiments, both agentscan be in solution. In another embodiment, the agents may be in solubleform, and then cross-linked to a surface, such as a cell expressing Fcreceptors or an antibody or other binding agent which will bind to theagents. In this regard, see for example, U.S. Patent ApplicationPublication Nos. 20040101519 and 20060034810 for artificial antigenpresenting cells (aAPCs) that are contemplated for use in activating andexpanding T cells in the present disclosure.

In one embodiment, the two agents are immobilized on beads, either onthe same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By wayof example, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the co-stimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one embodiment, a 1:1ratio of each antibody bound to the beads for CD4⁺ T cell expansion andT cell growth is used. In certain aspects of the present disclosure, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular embodiment an increaseof from about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present disclosure, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain embodiments ofthe disclosure, the ratio of anti CD28 antibody to anti CD3 antibodybound to the beads is greater than 2:1. In one particular embodiment, a1:100 CD3:CD28 ratio of antibody bound to beads is used. In anotherembodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. Ina further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beadsis used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody boundto beads is used. In one preferred embodiment, a 1:10 CD3:CD28 ratio ofantibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28ratio of antibody bound to the beads is used. In yet another embodiment,a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain embodiments the ratio of cellsto particles ranges from 1:100 to 100:1 and any integer valuesin-between and in further embodiments the ratio comprises 1:9 to 9:1 andany integer values in between, can also be used to stimulate T cells.The ratio of anti-CD3- and anti-CD28-coupled particles to T cells thatresult in T cell stimulation can vary as noted above, however certainpreferred values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, and 15:1 with one preferred ratio being at least 1:1particles per T cell. In one embodiment, a ratio of particles to cellsof 1:1 or less is used. In one particular embodiment, a preferredparticle: cell ratio is 1:5. In further embodiments, the ratio ofparticles to cells can be varied depending on the day of stimulation.For example, in one embodiment, the ratio of particles to cells is from1:1 to 10:1 on the first day and additional particles are added to thecells every day or every other day thereafter for up to 10 days, atfinal ratios of from 1:1 to 1:10 (based on cell counts on the day ofaddition). In one particular embodiment, the ratio of particles to cellsis 1:1 on the first day of stimulation and adjusted to 1:5 on the thirdand fifth days of stimulation. In another embodiment, particles areadded on a daily or every other day basis to a final ratio of 1:1 on thefirst day, and 1:5 on the third and fifth days of stimulation. Inanother embodiment, the ratio of particles to cells is 2:1 on the firstday of stimulation and adjusted to 1:10 on the third and fifth days ofstimulation. In another embodiment, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent disclosure. In particular, ratios will vary depending onparticle size and on cell size and type.

In further embodiments of the present disclosure, the cells, such as Tcells, are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. In a furtherembodiment, the beads and cells are first concentrated by application ofa force, such as a magnetic force, resulting in increased ligation ofcell surface markers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one embodiment the cells (for example,10⁴ to 10⁹T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer,preferably PBS (without divalent cations such as, calcium andmagnesium). Again, those of ordinary skill in the art can readilyappreciate any cell concentration may be used. For example, the targetcell may be very rare in the sample and comprise only 0.01% of thesample or the entire sample (i.e., 100%) may comprise the target cell ofinterest. Accordingly, any cell number is within the context of thepresent disclosure. In certain embodiments, it may be desirable tosignificantly decrease the volume in which particles and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and particles. For example, in one embodiment, aconcentration of about 2 billion cells/ml is used. In anotherembodiment, greater than 100 million cells/ml is used. In a furtherembodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45,or 50 million cells/ml is used. In yet another embodiment, aconcentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mlis used. In further embodiments, concentrations of 125 or 150 millioncells/ml can be used. Using high concentrations can result in increasedcell yield, cell activation, and cell expansion. Further, use of highcell concentrations allows more efficient capture of cells that mayweakly express target antigens of interest, such as CD28-negative Tcells. Such populations of cells may have therapeutic value and would bedesirable to obtain in certain embodiments. For example, using highconcentration of cells allows more efficient selection of CD8+ T cellsthat normally have weaker CD28 expression.

In one embodiment of the present disclosure, the mixture may be culturedfor several hours (about 3 hours) to about 14 days or any hourly integervalue in between. In another embodiment, the mixture may be cultured for21 days. In one embodiment of the disclosure the beads and the T cellsare cultured together for about eight days. In another embodiment, thebeads and T cells are cultured together for 2-3 days. Several cycles ofstimulation may also be desired such that culture time of T cells can be60 days or more. Conditions appropriate for T cell culture include anappropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or,X-vivo 15, (Lonza)) that may contain factors necessary for proliferationand viability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12,IL-15, TGFβ, and TNF-α or any other additives for the growth of cellsknown to the skilled artisan. Other additives for the growth of cellsinclude, but are not limited to, surfactant, plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanol. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo20, Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (T_(H), CD4⁺) that is greater than the cytotoxic orsuppressor T cell population (T_(C), CD8⁺). Ex vivo expansion of T cellsby stimulating CD3 and CD28 receptors produces a population of T cellsthat prior to about days 8-9 consists predominately of T_(H) cells,while after about days 8-9, the population of T cells comprises anincreasingly greater population of Tc cells. Accordingly, depending onthe purpose of treatment, infusing a subject with a T cell populationcomprising predominately of T_(H) cells may be advantageous. Similarly,if an antigen-specific subset of Tc cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

6.6 Compositions

The anti-glyco-MUC1 antibodies and/or anti-glyco-MUC1 ADCs of thedisclosure may be in the form of compositions comprising theanti-glyco-MUC1 antibody and/or ADC and one or more carriers, excipientsand/or diluents. The compositions may be formulated for specific uses,such as for veterinary uses or pharmaceutical uses in humans. The formof the composition (e.g., dry powder, liquid formulation, etc.) and theexcipients, diluents and/or carriers used will depend upon the intendeduses of the antibody and/or ADC and, for therapeutic uses, the mode ofadministration.

For therapeutic uses, the compositions may be supplied as part of asterile, pharmaceutical composition that includes a pharmaceuticallyacceptable carrier. This composition can be in any suitable form(depending upon the desired method of administering it to a patient).The pharmaceutical composition can be administered to a patient by avariety of routes such as orally, transdermally, subcutaneously,intranasally, intravenously, intramuscularly, intratumorally,intrathecally, topically or locally. The most suitable route foradministration in any given case will depend on the particular antibodyand/or ADC, the subject, and the nature and severity of the disease andthe physical condition of the subject. Typically, the pharmaceuticalcomposition will be administered intravenously or subcutaneously.

Pharmaceutical compositions can be conveniently presented in unit dosageforms containing a predetermined amount of an anti-glyco-MUC1 antibodyand/or anti-glyco-MUC1 ADC of the disclosure per dose. The quantity ofantibody and/or ADC included in a unit dose will depend on the diseasebeing treated, as well as other factors as are well known in the art.Such unit dosages may be in the form of a lyophilized dry powdercontaining an amount of antibody and/or ADC suitable for a singleadministration, or in the form of a liquid. Dry powder unit dosage formsmay be packaged in a kit with a syringe, a suitable quantity of diluentand/or other components useful for administration. Unit dosages inliquid form may be conveniently supplied in the form of a syringepre-filled with a quantity of antibody and/or ADC suitable for a singleadministration.

The pharmaceutical compositions may also be supplied in bulk fromcontaining quantities of ADC suitable for multiple administrations.

Pharmaceutical compositions may be prepared for storage as lyophilizedformulations or aqueous solutions by mixing an antibody and/or ADChaving the desired degree of purity with optionalpharmaceutically-acceptable carriers, excipients or stabilizerstypically employed in the art (all of which are referred to herein as“carriers”), i.e., buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic detergents, antioxidants, and othermiscellaneous additives. See, Remington's Pharmaceutical Sciences, 16thedition (Osol, ed. 1980). Such additives should be nontoxic to therecipients at the dosages and concentrations employed.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They may be present at a wide variety ofconcentrations, but will typically be present in concentrations rangingfrom about 2 mM to about 50 mM. Suitable buffering agents for use withthe present disclosure include both organic and inorganic acids andsalts thereof such as citrate buffers (e.g., monosodium citrate-disodiumcitrate mixture, citric acid-trisodium citrate mixture, citricacid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additionally, phosphate buffers, histidinebuffers and trimethylamine salts such as Tris can be used.

Preservatives may be added to retard microbial growth, and can be addedin amounts ranging from about 0.2%-1% (w/v). Suitable preservatives foruse with the present disclosure include phenol, benzyl alcohol,meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzylammonium chloride, benzalconium halides (e.g., chloride, bromide, andiodide), hexamethonium chloride, and alkyl parabens such as methyl orpropyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.Isotonicifiers sometimes known as “stabilizers” can be added to ensureisotonicity of liquid compositions of the present disclosure and includepolyhydric sugar alcohols, for example trihydric or higher sugaralcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol andmannitol. Stabilizers refer to a broad category of excipients which canrange in function from a bulking agent to an additive which solubilizesthe therapeutic agent or helps to prevent denaturation or adherence tothe container wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglyceroland sodium thio sulfate; low molecular weight polypeptides (e.g.,peptides of 10 residues or fewer); proteins such as human serum albumin,bovine serum albumin, gelatin or immunoglobulins; hydrophylic polymers,such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose,fructose, glucose; disaccharides such as lactose, maltose, sucrose andtrehalose; and trisaccacharides such as raffinose; and polysaccharidessuch as dextran. Stabilizers may be present in amounts ranging from 0.5to 10 wt % per wt of ADC.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe added to help solubilize the glycoprotein as well as to protect theglycoprotein against agitation-induced aggregation, which also permitsthe formulation to be exposed to shear surface stressed without causingdenaturation of the protein. Suitable non-ionic surfactants includepolysorbates (20, 80, etc.), polyoxamers (184, 188 etc.), and pluronicpolyols. Non-ionic surfactants may be present in a range of about 0.05mg/mL to about 1.0 mg/mL, for example about 0.07 mg/mL to about 0.2mg/mL.

Additional miscellaneous excipients include bulking agents (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E), and cosolvents.

6.7 Methods of Use

The anti-glyco-MUC1 antibody or binding fragments described herein canbe used in various diagnostic assays. For example, the antibodies andbinding fragments can be employed in immunoassays, such as competitivebinding assays, direct and indirect sandwich assays, andimmunoprecipitation assays, including immunohistochemistry,enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cellsorting (FACS), and Western blots.

The anti-glyco-MUC1 antibody or binding fragments described herein alsoare useful for radiographic in vivo imaging, wherein an antibody labeledwith a detectable moiety such as a radio-opaque agent or radioisotope isadministered to a subject, preferably into the bloodstream, and thepresence and location of the labeled antibody in the host is assayed.This imaging technique is useful in the staging and treatment ofmalignancies.

The anti-glyco-MUC1 antibody or binding fragments, ADCs and CARsdescribed herein are useful for treatment of glyco-MUC1 expressingcancers, particularly epithelial cancers such as breast cancer, ovariancancer, pancreatic cancer, and lung cancer.

When using the CARs of the disclosure for therapy, the therapeuticmethods of the disclosure comprising administering to a subject with aglyco-MUC1-expressing tumor an effective amount of a geneticallymodified cell engineered to express a CAR of the disclosure, for exampleas described in Section 6.4 or in embodiment 97 or embodiment 98.Methods of modifying cells, particularly T cells, to express a CAR, aredescribed in Section 6.5.1.

7. EXAMPLES 7.1 Example 1: Identification Of Anti-Glyco-Muc1 Antibodies

7.1.1. Overview

Chemoenzymatic synthesis of multiple-repeat MUC1 glycopeptides withdifferent O-glycan density and Tn (GalNAcα1-P-Ser/Thr) glycoforms wasdeveloped using recombinant glycosyltransferases. Different polypeptideGalNAc-transferase isoforms were used to direct sites of O-glycanoccupancy (Bennett et al., 1998). The optimal vaccine design was foundto be Tn glycoforms with high O-glycan density, and glycopeptidesconjugated to KLH were found to overcome tolerance. In wild-type Balb/cmice, the glycopeptides with complete O-glycan occupancy elicited thestrongest antibody response reacting with MUC1 expressed in breastcancer cell lines, thus representing the most effective vaccine design.The elicited humoral immune response showed remarkable specificity forcancer cells.

7.1.2. Materials and Methods

7.1.2.1 Chemoenzymatic Synthesis of Multimeric Tn MUC1 Glycopeptides

MUC1 60-mer (VTSAPDTRPAPGSTAPPAHG)n=3 (SEQ ID NO:47) peptide wassynthesized, as originally reported by Fontenot et al., 1993. Controlpeptides used were derived from tandem repeats (TRs) of MUC2(PTTTPISTTTMVTPTPTPTC) (SEQ ID NO:51) and MUC4 (CPLPVTDTSSASTGHATPLPV)(SEQ ID NO:52). Peptides were glycosylated in vitro using purifiedrecombinant human glycosyltransferases polypeptides GalNAc-T2,GalNAc-T4, and GalNAc-T1 (Bennett et al., 1998; Schwientek et al., 2002)as described in U.S. Pat. No. 6,465,220. GalNAc glycosylation of thepeptides was performed in a reaction mixture (1 mg peptide/mL)containing 25 mM cacodylate buffer (pH 7.4), 10 mM MnCl2, 0.25% TritonX-100, and 2 mM UDP-GalNAc. Glycosylation of 1 mg 60-mer peptide withtwo GalNAc per TR (MUC160Tn6) was obtained using GalNAc-T1.Incorporation of three GalNAc per TR (MUC160Tn9) was obtained usingGalNAc-T2. Substitution of all five putative O-glycosylation sites inthe MUC1 TR (MUC160Tn15) was performed using MUC160Tn9 as substrate in areaction with GalNAc-T4. Glycosylation was monitored using nano-scalereversed-phase columns (Poros R3, PerSeptive Biosystems, Framingham,Mass.) and MALDI-TOF mass spectrometry. The glycopeptides were purifiedby high-performance liquid chromatography (HPLC) on a Zorbax 300SB-C3column (9.4 mm×25 cm) (Agilent Technologies, Palo Alto, Calif.) in an1100 Hewlett Packard system (Avondale, Pa.) using 0.1% trifluoroaceticacid (TFA) and a gradient of 0-80% acetonitrile. Quantification andestimation of yields of glycosylation reactions were performed bycomparison of HPLC peaks by UV 210 absorbance using 10 μg weighedpeptide as standard. GalNAc glycosylation of peptides generally yielded80-90% recovery. Purified glycopeptides were characterized by MALDI-TOFmass spectrometry on a Voyager DE or Voyager DE Pro MALDI-TOF massspectrometer (PerSeptive Biosystems) equipped with delayed extraction.The MALDI matrix was 2,5-dihydroxybenzoic acid 10 g/L (Aldrich,Milwaukee, WI) dissolved in 2:1 mixture of 0.1% TFA in 30% aqueousacetonitrile. Samples dissolved in 0.1% TFA to a concentration of ˜1μmol/uL were prepared for analysis by placing 1 μL of sample solution ona probe tip followed by 1 uL of matrix. All mass spectra were obtainedin the linear mode. Data processing was carried out using GRAMS/386software (Galactic Industries, Salem, N.H.).

7.1.2.2 Immunization Protocol

Glycopeptides were coupled to KLH (Pierce, Rockford, Ill.) usingglutaraldehyde. Efficiency of conjugation was assessed by analyzing thereaction by size exclusion chromatography on a PD-10 column usinganti-MUC1 ELISA of fractions. Essentially all reactivity was found withthe excluded fraction and insignificant reactivity in the includedfractions expected to contain peptides. Further evaluation includedcomparative titration analysis of the KLH conjugate with thecorresponding glycopeptide in ELISA. Both analyses indicated that theconjugation was near complete, which should result in a KLH toglycopeptide ratio of 1:300. Female Balb/c wild-type mice were injectedsubcutaneously with 10 or 15 μg of (glyco)peptide in a total volume of200 uL (1:1 mix with Freunds adjuvant, Sigma). Mice received fourimmunizations 14 days apart, and blood samples were obtained by tail oreye bleeding 1 week following the third and fourth immunization.

7.1.2.3 Generation of Mouse MAb anti-Tn-MUC1

A MAb was produced, from a wild-type Balb/c mouse immunized with thefully GalNAc-glycosylated 60-mer MUC1 glycopeptide coupled to KLH.Screening was based on glycopeptide ELISA followed by immunocytologywith breast cancer cell lines (MCF7 and T47D) and immunohistology withcancer tissues. Selection was based on reactivity pattern similar tototal sera of the same mouse.

7.1.2.4 ELISA

ELISA were performed using 96-well MaxiSorp plates (Nunc, Denmark).Plates were coated overnight at 4° C. with 1 μg/mL of glycopeptides inbicarbonate-carbonate buffer (pH 9.6), blocked with 5% bovine serumalbumin (BSA) in phosphate-buffered saline (PBS), followed by incubationwith sera (diluted in PBS) or MAbs for 2 h at room temperature. Boundantibodies were detected with peroxidase-conjugated rabbit anti-mouseimmunoglobulins (DakoCytomation, Glostrup, Denmark) or isotype-specificantibodies peroxidase-conjugated goat anti-mouse IgM, IgG1, IgG2a,IgG2b, or IgG3 (Southern Biotechnology Associates, Birmingham, Ala.).Plates were developed with O-phenylenediamine tablets (DakoCytomation)and read at 492 nm. Control antibodies included anti-MUC1 antibodiesHMFG2 and SM3 (Burchell et al., 1987) and anticarbohydrate antibodies5F4 (Tn) and 3F1 (STn) (Mandel et al., 1991). Control sera included miceimmunized with MUC4 mucin peptide linked to KLH.

7.1.3. Results

Glycopeptide specific mAbs were produced to GalNAc-MUC1 usingGalNAc-MUC1 60-mer glycopeptide conjugated to KLH as immunogen. Using anELISA assay, the generated mAb GO2 (5F7) reacted specifically with theMUC1 tandem repeat (VTSAPDTRPAPGSTAPPAHG)₃ (SEQ ID NO:47) that has beenglycosylated in vitro using purified recombinant humanglycosyltransferases GalNAc-T1, GalNAc-T2, and GalNAc-T4, with noreaction with the corresponding MUC1 peptide without GalNAc-residues orirrelevant glycopeptides with the same type of Tn glycoform. Results ofthe ELISA assay are shown in FIG. 1.

7.2 Example 2: Characterization of Anti-Glyco-Muc1 Antibodies 7.2.1.Overview

Monoclonal antibody GO2 (5F7) was characterized for the specificity ofits binding to the Tn glycoforms of MUC1 associated with cancer cells.

7.2.2. Materials and Methods

7.2.2.1 Immunocytochemistry

Cell lines were fixed for 10 min in ice-cold acetone or inmethanol:acetone. Fixed cells were incubated overnight at 5° C. withmouse sera (1:200/1:400/1:800) or MAbs, followed by incubation for 45min at room temperature with fluorescein isothiocyanate(FITC)-conjugated rabbit anti-mouse immunoglobulins (DakoCytomation).Slides were mounted in glycerol containing p-phenylenediamine andexamined in a Zeiss fluorescence microscope (FluoresScience,Hallbergmoos, Germany).

7.2.2.2 Immunohistochemistry

Formalin fixed, paraffin wax embedded tissues of breast carcinoma wereobtained. All cases were conventionally classified by histological type.The avidin-biotin peroxidase complex method was used for immunostaining.Paraffin sections were dewaxed, rehydrated, and treated with 0.5% H₂O₂in methanol for 30 min. Sections were rinsed in TBS and incubated for 20min with rabbit non-immune serum. Sections were rinsed and incubatedovernight at 5° C. with primary antibody. Sections were rinsed andincubated with biotin-labeled rabbit anti-mouse serum (DakoCytomation)diluted 1:200 in TBS for 30 min, rinsed with TBS, and incubated for 1 hwith avidin-biotin peroxidase complex (DakoCyto-mation). Sections wererinsed with TBS and developed with 0.05% 3,3′-diaminobenzidinetetrahydrochloride freshly prepared in 0.05 M TBS containing 0.1% H₂O₂.Sections were stained with hematoxylin, dehydrated, and mounted.

7.2.3. Results

Immunohistochemistry of colorectal carcinoma, pancreatic carcinoma, andinvasive breast adenocarcimas were performed with GO2. Staining ofcolorectal cancer tissue (FIG. 2) demonstrated strong labeling ofintracellular and surface structures on a large proportion of the cancercells. In contrast no or significantly lower reactivity was seen tohealthy columnar epithelial cells. The labeling in healthy cells wasrestricted to intracellular structures, which is expected due to thepresence of large amounts of biosynthetic intermediates (GalNAc-modifiedglycoproteins) in cells with high secretory capacity such as coloniccumnar epithelia. A similar pattern was observed with pancreatic (FIG.3) and breast cancer tissue (FIG. 4) with strong reactivity with cancercells and none or limited reactivity with intracellular structures insurrounding healthy epithelia or connective tissue cells.

7.3 Example 3: Sequence Analysis of Anti-Glyco-Muc1 Antibodies

mRNA from the hybridoma producing monoclonal antibody GO2 (5F7) wasprepared, reverse transcribed and sequenced.

The nucleotide sequences encoding the heavy and light chain variableregions with their signal sequences are set forth in SEQ ID NO:11 andSEQ ID NO:12, respectively. The heavy and light chain variable regionsencoded by SEQ ID NO:11 and SEQ ID NO:12 are set forth in SEQ ID NO:1and SEQ ID NO:2, respectively. The predicted mature variable regionsequences (following truncation of the signal peptide) are set forth inSEQ ID NO:3 and SEQ ID NO:4, respectively, and are encoded by SEQ IDNO:13 and SEQ ID NO:14, respectively. The predicted heavy chain CDRsequences (IMGT definition) are set forth in SEQ ID NOs:5-7,respectively, and the predicted light chain CDR sequences (IMGTdefinition) are set forth in SEQ ID NOs:8-10, respectively.

7.4 Example 4: Drug Delivery to Cancer Cells with Anti-Glyco-Muc1Antibodies

7.4.1. Overview

Monoclonal antibody GO2 (5F7) was tested for its ability to deliver acytotoxic agent to target cells.

7.4.2. Materials and Methods

OVCar human ovarian carcinoma cells were added to a 24-well cell cultureplate at 1,000 cells/well. Monoclonal antibody GO2 and a secondaryantibody conjugated to the antitubulin agent monomethyl auristatin F(MMAF) (anti-mFc-NC-MMAF) (Moradec catalog no. AM-101-AF) were added tothe plate to give the following concentrations (in pg/ml) of GO2 andADC:

TABLE 2 Column Row 1 2 3 4 5 6 A GO2: 5 GO2: 1 GO2: 0.2 GO2: 0.04 GO2:0.008 GO2: 0 ADC: 2.0 ADC: 2.0 ADC: 2.0 ADC: 2.0 ADC: 2.0 ADC: 2.0 BGO2: 5 GO2: 1 GO2: 0.2 GO2: 0.04 GO2: 0.008 GO2: 0 ADC: 0.6 ADC: 0.6ADC: 0.6 ADC: 0.6 ADC: 0.6 ADC: 0.6 C GO2: 5 GO2: 1 GO2: 0.2 GO2: 0.04GO2: 0.008 GO2: 0 ADC: 0.2 ADC: 0.2 ADC: 0.2 ADC: 0.2 ADC: 0.2 ADC: 0.2D GO2: 5 GO2: 1 GO2: 0.2 GO2: 0.04 GO2: 0.008 GO2: 0 ADC: 0 ADC: 0 ADC:0 ADC: 0 ADC: 0 ADC: 0

The plate was incubated at 37° C. for 48 hours. After the 48 hourincubation, AlamarBlue® (Invitrogen) was added to each well, andfluorescence at 600 nm measured.

7.4.3. Results

Results are shown in FIG. 5. The results show that the cellular toxicityis dependent on primary antibody (GO2) concentration and presence of theantibody, and secondary ADC conjugated antibody concentration andpresence. In other words, GO2 induces cellular toxicity of this cancercell line when coupled with a secondary antibody that carries acytotoxic agent MMAF.

7.5 Example 5: Circulating Tumor Cell Quantification with Anti-Muc1Antibodies

7.5.1. Overview

Monoclonal antibody GO2 (5F7) was tested for its ability to be used toquantify circulating tumor cells.

7.5.2. Materials and Methods

GO2 was conjugated to a magnetic separation bead and allowed to interactwith samples of different concentrations of tumor cells. Cells and beadswere pulled out of solution with a magnet and washed several times toremove unbound material. GO2 that was conjugated to horseradishperoxidase was then applied to the magnetic separation beads containingbound cancer cells, incubated, and then unbound conjugated GO2 waswashed away. A colorimetric reaction was performed using TNB as asubstrate. The reaction was terminated with sulfuric acid and then OD450 readings were taken on the samples.

7.5.3. Results

Results are shown in FIG. 6. The results of the assay demonstrated GO2binding of tumor cells.

7.6 Example 6: Immunohistochemical Staining of Tumor Tissue UsingAnti-Glyco-Muc1 Antibodies

7.6.1. Materials and Methods

Sections from six formalin fixed, paraffin embedded (FFPE) tissue microarrays (TMAs) were cut at 2.5 μm thickness. TMAs from breast cancer(BC), colorectal cancer (CRC), ovarian cancer (OVC), non-small cell lungcancer (NSCLC) and prostate cancer (PrC) tumors were used in the study.25-47 tumor tissue cores per TMA were available for evaluation. Coresize was 1 mm, 2 mm or 3 mm, depending on the TMA. Each tissue corerepresented one patient.

Staining was performed on a Discovery XT autostainer (Ventana MedicalSystems). Following antigen retrieval with cell condition 1 (CC1)solution (Ventana Medical Systems), the GO2 was applied at aconcentration of 1 μg/mL in Dako green medium antibody diluent andincubated for 60 min at 37° C. Binding of GO2 to tumor cells wasdetected using the Optiview DAB IHC detection kit (Ventana MedicalSystems) visualized in DAB (brown precipitate).

7.6.2. Results

Representative images of MUC1 positive TMA tumor cores are shown in FIG.7. In BC and OVC TMAs, the majority of spots (>90%) showed moderate orstrong binding of GO2 to tumor cells. 70% and 51% of NSCLC and CRC casesshowed moderate and strong binding of the antibody to tumor cells,respectively. In prostate cancer, the antigen appeared to be lessexpressed. Only 28% of the spots in the TMA 1 revealed a moderate orstrong staining intensity when applying GO-2. Staining patterns werealways cytoplasmic and in many cases membrane-bound. An apical membranestaining pattern was observed in few cores.

7.7 Example 7: Production and Purification of an Anti-MUC1 Antibody inT-Cell Bispecific (TCB) Format

7.7.1. Materials and Methods

7.7.1.1 Expression Vector Production

The GO2 antibody was converted into TCB format, includingknob-into-holes and P329G/L234A/L235A (“PGLALA”) mutations in the Fcregion and charged residues in the MUC1 CH1 (147E/213E; “EE”) and CL(123R/124K; “RK”) regions (see SEQ ID NOs: 43-46). The TCB antibody isillustrated in FIG. 8. Briefly, the variable heavy and variable lightchains of GO2 mAb were synthesized (Geneart, Regensburg, Germany) andinserted into suitable expression vectors in which they are fused to theappropriate human constant heavy or human constant light chains. Theexpression cassettes in these vectors consist of the CMV promoter,Intron A with 5′ UTR and a BGH polyadenylation site. In addition, theplasmids contain the oriP region from the Epstein Barr virus for thestable maintenance in HEK293 cells harboring the EBV nuclear antigen(EBNA).

7.7.1.2 Transient Transfection and Production

The antibodies were transiently produced in HEK293 EBNA cells using aPEI mediated transfection procedure as follows. HEK293 EBNA cells arecultivated in suspension serum free in Excell culture medium, containing6 mM L-Glutamine. For the production of antibodies in a 500 ml shakeflask, 300 million HEK293 EBNA cells are seeded 24 hours beforetransfection (for alternative scales all amounts were adjustedaccordingly). For transfection, cells are centrifuged for 10 min at210×g and the supernatant is replaced by pre-warmed 20 ml CD CHO medium.Expression vectors are mixed in 20 ml CD CHO medium to a final amount of200 μg DNA. After addition of 540 μl PEI, solution is vortexed for 15 sand subsequently incubated for 10 min at room temperature. Afterwardscells are mixed with the DNA/PEI solution, transferred to a 500 ml shakeflask and incubated for 3 hours by 37° C. in an incubator with a 5% CO₂atmosphere. After incubation, 160 ml Ex-cell® medium (Sigma-Aldrich),containing 6 mM glutamine, 1.25 mM valproic acid and 12.5% Pepsoy, isadded and cells are cultivated for 24 hours. One day after transfection,12% Feed 7 (48 mL)+3 g/L glucose is added. After 7 days, cultivationsupernatant is collected for purification by centrifugation for 45 minat 3000×g. The solution is sterile filtered (0.22 μm filter) and sodiumazide in a final concentration of 0.01% w/v is added. The solution isthen stored at 4° C.

7.7.1.3 Antibody Purification

Secreted proteins were purified by affinity chromatography using ProteinA, followed by size exclusion chromatography. For affinitychromatography, the supernatant was loaded on a Protein A MabSelect SuRecolumn (GE Healthcare) equilibrated with 20 mM sodium phosphate, 20 mMsodium citrate pH 7.5. Unbound protein was removed by washing withequilibration buffer. The bound protein was eluted using either a step(standard IgG) or a gradient (bispecific antibody) elution created with20 mM sodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3.0.The pH of collected fractions was adjusted by adding 1/10 (v/v) of 0.5 Msodium phosphate pH8.0. The protein was concentrated and filtered priorto loading on a HiLoad Superdex 200 column (GE Healthcare) equilibratedwith 20 mM histidine, 140 mM sodium chloride, pH 6.0.

The aggregate content of eluted fractions was analyzed by analyticalsize exclusion chromatography. Therefore, 30 μl of each fraction wasapplied to a TSK G3000 SWXL column (TOSOH, 7.8 mm×30 cm) equilibratedwith 200 mM Arginine, 25 mM K₂PO₄, 125 mM Sodium chloride, 0.02% NaN₃,pH 6.7. Fractions containing less than 2% oligomers were pooled andconcentrated to final concentration of 1-1.5 mg/ml using centrifugalconcentrators (Millipore, Amicon® ULTRA—15, 30k MWCO). Purified proteinswere stored at −80° C.

7.7.2. Results

Production yield and quality of GO2 TCB antibodies are shown in Table 5.

TABLE 5 Yield Monomer HMW LMW Purity [%] Molecule [mg/L] [%] SEC [%] [%]CE-SDS GO2 TCB 0.51 94.16 0.00 5.84 85.61

7.8 Example 8: Jurkat-NFAT Reporter Assay to Monitor Target ExpressionEx Vivo in Undigested Patient-Derived Tumor Samples

7.8.1. Overview

A Jurkat NFAT reporter assay was used to monitor target expression(glyco-MUC1) ex vivo in undigested primary human tumor samples using aGO2 TCB.

7.8.2. Materials and Methods

7.8.2.1 Materials

-   -   GO2 TCB (see Example 7)    -   DP47 TCB (non-targeted, negative control)    -   Matrigel (Item no. 734-1101, Corning/VWR, Switzerland)    -   Corning® Costar® Ultra-Low attachment multiwell plates (Item no.        CLS7007-24EA, Sigma)    -   Cell culture microplate, 96 well (Item no. 655098, Greiner        Bio-one, Switzerland)    -   RPMI1640 Medium (Item no. 42401-018, FisherScientific, Schweiz)    -   Jurkat Medium: RPM11640 Medium with 2 g/l D-Glucose, 2 g/l        NaHCO₃, 10% FCS, 25 mM HEPES, 2 mM L-Glutamine, 1×NEAA,        1×Sodium-Pyruvate, 200 μg/ml Hygromycine B    -   Jurkat NFAT luciferase reporter cells (Promega)    -   Tumor samples received from Indivumed GmbH, Germany. Samples        were shipped over night in transport medium. About 24 h after        surgery the samples were cut in small pieces.

7.8.2.2 Methods

96-well cell culture microplates were prepared by adding 17 μl of coldmatrigel. The plate was incubated for 2 min at 37° C. before tumorpieces were added (triplicates). 33 μl of cold matrigel was added perwell and the plate was incubated again for 2 min at 37° C. 100 μl (50 nMor 5 nM) of TCB antibody dilution (diluted in Jurkat medium withoutHygromycine but with 2× Penicillin/Streptomycine) was added per well.Jurkat-NFAT reporter cells were harvested and viability was assessedusing ViCell. Cells were centrifuged at 350×g for 7 min before they wereresuspended in Jurkat medium without Hygromycine. 50 μl of the cellsuspension was added per well (50,000 cells/well). The plate wasincubated for 4 to 5 h at 37° C. in a humidified incubator before it wastaken out for luminescence read out. 50 μl of ONE-Glo solution was addedto each well and incubated for 10 min at room temperature in the dark.Luminescence was detected using WALLAC Victor3 ELISA reader(PerkinElmer2030), with a 5 sec/well detection time.

7.8.3. Results

Results from tumor samples from three patients are shown in FIGS. 9-11.The results shown in FIG. 9 are from tumor samples obtained from apatient having a malignant neoplasm of bronchus and lung: middle lobe,bronchus or lung, squamous cell carcinoma. The results shown in FIG. 10are from tumor samples obtained from a patient having a malignantneoplasm of bronchus and lung: lower lobe, bronchus or lung,non-keratinizing squamous cell carcinoma. The results shown in FIG. 11are from tumor samples obtained from a patient having a malignantneoplasm of bronchus and lung: upper lobe, bronchus or lung,adenocarcinoma with acinar type. Each bar in FIGS. 9-11 represents themean of triplicates. Standard error is indicated by error bars. Thedotted line indicates luminescence for Jurkat NFAT cells incubated withtumor samples without any TCB. Two-tailed, unpaired t-test was used forstatistical analysis. P-values below 0.05 were considered as significantand were indicated with stars (* P≤0.05; ** P≤0.001; *** P≤0.001). Ineach of FIGS. 9-11, tumor samples incubated with GO2 TCB displayedsignificantly more luminescence than samples incubated with DP47negative control TCB.

7.9 Example 9: In Vitro Characterization of GO2 TCB

7.9.1. Overview

GO2 TCB (Example 7) recognizing the tumor-specific aberrantlyglycosylated MUC1 was functionally characterized on tumor cellsexpressing MUC1.

7.9.2. Materials and Methods

7.9.2.1 Cell Lines and PBMCs

T3M4 pfzv and MCF7 cs engineered tumor cell lines were cultured in DMEMwith 10% FCS and 2 mM Glutamine. MCF10A is a human non-tumorigenicmammary epithelial cell line (ATCC® CRL-10317). HBEpiC are humanbronchial epithelial cells (Sciencell #3210). PBMCs were isolated bygradient centrifugation using whole blood from healthy volunteers.

7.9.2.2 Target Binding by Flow Cytometry

Target cells as indicated were harvested with Cell Dissociation Buffer,washed with PBS and resuspended in FACS buffer. The antibody stainingwas performed in a 96-well round bottom plate. 200,000 cells were seededper well. The plate was centrifuged for 4 min at 400 g and thesupernatant was removed. The test antibodies were diluted in FACS bufferand 30 μl of the antibody solution was added to the cells for 30 min at4° C. To remove unbound antibody the cells were washed twice with FACSbuffer before addition of the diluted secondary antibody (PE-conjugatedAffiniPure F(ab′)2 Fragment goat anti-human IgG Fcγ Fragment Specific,Jackson ImmunoResearch #109-116-170). After 30 min incubation at 4° C.unbound secondary antibody was washed away. Before measurement the cellswere resuspended in 200 μl FACS buffer and analyzed by flow cytometryusing BD Fortessa. Assays were performed in triplicates.

7.9.2.3 T Cell Mediated Tumor Cell Killing and T Cell Activation

Target cells were harvested with Trypsin/EDTA, counted and viability waschecked. The cells were resuspended in their respective medium with afinal concentration of 300,000 cells per ml. Then 100 μl of the targetcell suspension was transferred into each well of a 96-flat bottomplate. The plate was incubated overnight at 37° C. in the incubator toallow adherence of the cells to the plate. On the next day PBMCs wereisolated from whole blood. The blood was diluted 2:1 with PBS andoverlayed on 15 ml Histopaque-1077 (#10771, Sigma-Aldrich) in Leucoseptubes and centrifuged for 30 min at 450 g without brake. Aftercentrifugation, the band containing the cells was collected with a 10 mlpipette and transferred into 50 ml tubes. The tubes were filled up withPBS until 50 ml and centrifuged (400 g, 10 min, room temperature). Thesupernatant was removed and the pellet resuspended in PBS. Aftercentrifugation (300 g, 10 min, room temperature), supernatants werediscarded, 2 tubes were pooled and the washing step was repeated (thistime centrifugation 350 g, 10 min, room temperature). Afterwards, thecells were resuspended and the pellets pooled in 50 ml PBS for cellcounting. After counting cells were centrifuged (350 g, 10 min, roomtemperature) and resuspended at 6 million cells per ml in RPMI with 2%FCS and 2 nM Glutamine. Medium was removed from plated target cells andthe test antibodies diluted in RPMI with 2% FCS and 2 nM Glutamine wereadded. 300,000 cells of the effector cell solution were transferred toeach well resulting in a E:T ratio of 10:1. To determine the maximalrelease target cells were lysed with Triton X-100. LDH release wasdetermined after 24 h and 48 h using Cytotoxicity Detection Kit(1644793, Roche Applied Science). Activation marker upregulation on Tcells after tumor cell killing was measured by flow cytometry. Briefly,PBMCs were harvested, transferred into a 96-well round bottom plate andstained with CD4 APC (300514, BioLegend), CD8 FITC (344704, BioLegend),CD25 BV421 (302630, BioLegend), CD69 PE (310906, BioLegend) antibodiesdiluted in FACS buffer. After 30 min incubation at 4° C. the cells werewashed twice with FACS buffer. Before measuring the fluorescence usingBD Fortessa II the cells were resuspended in 200 μl FACS buffer. Assayswere performed in triplicates.

7.9.2.4 Cytokine/Chemokine Release by Cytometric Bead Array

Cytokine/chemokine secretion in the supernatant was measured by flowcytometry, using the cytometric bead array (CBA), according to themanufacturer's guidelines. Supernatants from T cell mediated killingassays were collected and stored at −20° C. Supernatants weresubsequently thawed and tested according to manufacturer's instructions.The following CBA kits (BD Biosciences) were used: CBA human interferongamma (IFNγ) Flex Set (E7), CBA human Granzyme B Flex Set (D7), CBAhuman IL6 Flex Set (A7), CBA human IL8 Flex Set (A9), CBA human IL10Flex Set (B7) and CBA human tumor necrosis factor (TNF) Flex Set (D9).Samples were measured using the BD FACS Canto II and analyses wereperformed using the Diva Software (BD Biosciences). Assays wereperformed in triplicates.

7.9.3. Results

Binding of GO2 TCB to the breast cancer cell line MCF7 and thepancreatic cancer cell line T3M4, both engineered to express theaberrantly glycosylated MUC1, was confirmed (FIG. 12). Subsequently,activity of GO2 TCB was tested on both tumor cell lines, MCF7 and T3M4,using freshly insolated PBMCs (FIG. 13 and FIG. 14). Tumor cell killingof both cell lines was detected after 24 h and even stronger after 48 h.This was accompanied by strong activation of CD4 T cells and CD8 T cellsdetermined by upregulation of the two activation markers CD25 and CD69and the release of IL6, IL8, IL10, IFNγ, TNFα and Granzyme B into thesupernatant. As negative control the respective untargeted TCB wasincluded.

To prove that GO2 TCB does not bind to normally glycosylated MUC1 onepithelial cells, binding to MCF10A, which is a human non-tumorigenicmammary epithelial cell line, and to HBEpiC, which are primary humanbronchial epithelial cells, was tested. As a positive control the HMFG1TCB, which does not discriminate between MUC1 expressed on normal and ontumor cells, was included. HFMG1 TCB was found to bind to both testedcells, confirming the expression of MUC1, but GO2 TCB was not able tobind to these cells (FIG. 15). In addition, GO2 TCB was tested to see ifit would induce killing or T cell activation in the presence of normalepithelial cells expressing MUC1. This was tested on MCF10A cells andthere was no killing or T cell activation detectable with GO2 TCB,whereas HMFG1 TCB induced killing as well as T cell activation (FIG.16).

7.10 Example 10: Functional Characterization of GO2 and GO2 TCBAntibodies by Surface Plasmon Resonance

7.10.1. Overview

GO2 and GO2 TCB (Example 7) were characterized by surface plasmonresonance.

7.10.2. Materials and Methods

7.10.2.1 Binding of GO2 and GO2 TCB to Immobilized Glycopeptides

Binding of the GO2 antibody and GO2 TCB to human and cynomolgusglycopeptides (Table 6) was assessed by surface plasmon resonance (SPR).All SPR experiments were performed on a Biacore T200 at 25° C. withHBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA,0.005% Surfactant P20, Biacore, Freiburg/Germany).

TABLE 6 Concentration  Glycopeptide Sequence in PBS Human peptidePDTSAAPGSTAPPAHVVTSAP 0.9 mg/ml (SEQ ID NO: 48) Cynomolgus PDTSAAPGSTGPPAHVVTSAP 1.8 mg/ml peptide (SEQ ID NO: 49)

The biotinylated glycopeptides were dissolved in PBS and the finalconcentration was between 0.9 and 1.8 mg/ml (Table 6). Biotinylatedglycopeptides were directly coupled to a flow cell of a streptavidin(SA) sensor chip. Immobilization levels up to 880 resonance units (RU)were used. The GO2 antibody or the GO2 TCB were injected with a flow of30 μL/minute through the flow cells, over 240 seconds and at aconcentration of 1000 nM (FIG. 17). The dissociation was monitored for500 sec. Bulk refractive index differences were corrected for bysubtracting the response obtained in a reference flow cell, where noprotein was immobilized.

7.10.2.2 Avidity of GO2 and GO2 TCB to Immobilized Glycopeptides

The avidity of GO2 and GO2 TCB was assessed by surface plasmon resonance(SPR). All SPR experiments were performed on a Biacore T200 at 25° C.with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mMEDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany). Biotinylatedglycopeptides (Table 6) were directly coupled to a flow cell of astreptavidin (SA) sensor chip. Immobilization levels up to 200 resonanceunits (RU) were used.

The GO2 antibody or the GO2 TCB were injected with a flow of 30μL/minute through the flow cells over 120 seconds and at a concentrationrange from 3.9 to 1000 nM (1:2 dilution). The dissociation was monitoredfor 400 sec. Bulk refractive index differences were corrected for bysubtracting the response obtained in a reference flow cell, where noprotein was immobilized. The KDs were derived, despite the bivalency ofthe interaction, by fitting the curve to a 1:1 Langmuir binding usingthe Biaeval software (GE Healthcare). The “apparent” KD can therefore beused for comparison purposes only.

7.10.3. Results

As can be seen in the sensorgrams of FIG. 18, GO2 antibody (FIG. 18A)and GO2 TCB (FIG. 18B) bind both human and cynomolgous glycopeptides.GO2 antibody and GO2 TCB bind with higher avidity to cynomolgus than tohuman glycopeptide.

As can be seen in FIG. 19, binding of a bivalent GO2 binder (IgG, TCB)to human glycopeptide is in the three-digit nanomolar (FIGS. 19A and19C), whereas binding to cynomolgus glycopeptide is in the two-digitnanomolar (FIGS. 19B and 19D).

7.11 Example 11: Exploratory Single Dose Pharmacokinetic andTolerability Study of GO2 TCB in Cynomolgus Monkeys

7.11.1. Overview

The objectives of this study are to determine the pharmacokinetics andtolerability of the GO2 TCB described in Example 7, when given by asingle intravenous injection to cynomolgus monkeys.

7.11.2. Materials and Methods

7.11.2.1 Preparation of GO2 TCB

Thawing of the frozen stock solution of GO2 TCB (2.12 mg/mL) andformulation buffer (20 mM Histidine, 140 mM NaCl, 0.01% Tween 20; pH6.0) is done overnight in a fridge set to maintain 4° C. Test itemdosing formulations are prepared under sterile conditions at appropriateconcentrations to meet dose level requirements by dilution withformulation buffer.

The dosing formulation is prepared within 2 hours before injection andstored at room temperature until use. Polypropylene containers are usedfor preparation and storage of dosing formulation to prevent adsorption.Dosing formulations should are not filtered, nor stirred or shaken. Anymixing is done either by gentle pipetting or gentle swinging of thecontainer.

7.11.2.2 Animals

Cynomolgus monkeys (Macaca fascicularis) 2-4 years of age and weighingless than 4 kg are used in the study. The animals are allowed toacclimate to the test facility primate toxicology accommodation for atleast 6 weeks before the commencement of dosing.

During the week before the commencement of dosing, the animals areapproved for entry into the experiment on the basis of a satisfactoryveterinary examination (performed shortly after arrival), clinicalobservation records, body weight profile and clinical pathologyinvestigations.

Animals selected for the study are randomly allocated to cages based onsupplied group compatibility information and then allotted individualstudy numbers. The animals are allocated a cage in groups of up to 5.

7.11.2.3 Husbandry

Animals are socially housed where possible, in groups of up to 5 by sexin two story gang pens measuring 1.61×1.66×2.5 m on the lower story and1.61×1.66×2.03 m on the upper. Bedding material is wood shavings. Thereare no known contaminants in the bedding that would interfere with theobjectives of the study.

The targeted conditions for animal room environment are be as follows:

-   -   Temperature: 18-24° C.    -   Humidity: 40-70%    -   Ventilation: a minimum of 10 air changes per hour    -   Light Cycle: 12 hours light and 12 hours dark (except when        interrupted by study procedures/activities).

There is automatic control of temperature and humidity which iscontinuously monitored and recorded. There is automatic control of lightcycle.

Special Diets Services (SDS) MP(E) Short SQC Diet is provided as a dailyration throughout the study. Approximately 200 gram ration of feed peranimal is provided once daily. There are no known contaminants in thefeed that would interfere with the objectives of the study. The animalshave access to water ad libitum from the public supply. There are noknown contaminants in the water that would interfere with the objectivesof the study.

The animal's home environment is enriched to promote social interaction,play and exploration. The animals have perches and materials such asplastic toys, balls, climbing frames and stainless steel mirrors. Theseare exchanged frequently to reduce familiarity. Prior to exchange, alltoys and climbing frames are thoroughly cleaned to avoidcross-contamination. The animals are also offered a range of othertreats such as forage mix, vegetables, nuts, biscuits and fruitsnormally on a daily basis.

Veterinary care is available throughout the course of the study andanimals are examined by the veterinary staff as warranted by clinicalsigns or other changes.

7.11.2.4 Experimental Design

One male and one female animal are administered GO2 TCB

TABLE 7 Dose Level Dose Volume Dose Concentration Group No. (μg/kg)(mL/kg) (μg/kg) 1 (1 male and 1 100 1 100 female) 2 (1 male and 1 300 1300 female)

GO2 TCB is administered to the appropriate animals by a singleintravenous slow bolus injection (1-2 min) in the saphenous vein or tailvein at least 8 days apart. is staggered so that only one male and onefemale receive a new dose level on any single day. Based on theobservations from the previous dose level (including clinical pathologydata), the doses are increased or decreased for the next dose group.Naïve animals are used for each dose level. The doses are given using asyringe with attached Vygon infusion needle.

Animals are necropsied ca 72 hours after dosing (after the lastscheduled sample has been taken). For all animals which have to beterminated prior to the scheduled date due to severe clinical symptoms,complete panel of clinical pathology (additional sampling prior totermination, if feasible) and histopathology are analyzed.

The intravenous injection route of administration has been selected forthis study as this route is a possible route of clinical application.The low dose and the high dose levels were chosen to cover a clinicallyrelevant dose range and to minimize the potential harm to the animals.The low dose was selected based on the experience in the cynomolgusmonkey with similar T-cell bispecific antibodies of similar potency andthe high-dose represents a 3-fold increment thereof.

7.11.3. Results

GO2 TCB is tolerated at the tested doses.

8. SPECIFIC EMBODIMENTS, CITATION OF REFERENCES

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the disclosure(s). The presentdisclosure is exemplified by the numbered embodiments set forth below.

1. An anti-glyco-MUC1 antibody or antigen binding fragment that:

-   -   a. preferentially binds to a glyco-MUC1 epitope that is        overexpressed on cancer cells as compared to normal cells; and    -   b. competes with an antibody or antigen binding fragment        comprising a heavy chain variable (VH) sequence of SEQ ID NO:3        and a light chain variable (VL) sequence of SEQ ID NO:4 for        binding to the breast cancer cell line MCF7 or T47D.

2. An anti-Glyco-MUC1 antibody or antigen binding fragment that

-   -   a. binds to the MUC1 tandem repeat (VTSAPDTRPAPGSTAPPAHG)3 that        has been glycosylated in vitro using purified recombinant human        glycosyltransferases GalNAc-T1, GalNAc-T2, and GalNAc-T4, and        (referred to hereinafter as the “first epitope”); and    -   b. competes with an antibody or antigen binding fragment        comprising a heavy chain variable (VH) sequence of SEQ ID NO:3        and a light chain variable (VL) sequence of SEQ ID NO:4 for        binding to the breast cancer cell line MCF7 or T47D.

3. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 1 or embodiment 2 comprising a complementarity determiningregion (CDR) H1 comprising the amino acid sequence of SEQ ID NO:33, aCDR-H2 comprising the amino acid sequence of SEQ ID NO:29, a CDR-H3comprising the amino acid sequence of SEQ ID NO:25, a CDR-L1 comprisingthe amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the aminoacid sequence of SEQ ID NO:9, and a CDR-L3 comprising the amino acidsequence of SEQ ID NO:31.

4. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 3, wherein CDR-H1 comprises the amino acid sequence of SEQ IDNO:5.

5. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 3, wherein CDR-H1 comprises the amino acid sequence of SEQ IDNO:23.

6. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 3, wherein CDR-H1 comprises the amino acid sequence of SEQ IDNO:28.

7. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 3, wherein CDR-H1 comprises the amino acid sequence of SEQ IDNO:32.

8. The anti-glyco-MUC1 antibody or antigen-binding fragment of any oneof embodiments 3 to 7, wherein CDR-H2 comprises the amino acid sequenceof SEQ ID NO:6.

9. The anti-glyco-MUC1 antibody or antigen-binding fragment of any oneof embodiments 3 to 7, wherein CDR-H2 comprises the amino acid sequenceof SEQ ID NO:24.

10. The anti-glyco-MUC1 antibody or antigen-binding fragment of any oneof embodiments 3 to 9, wherein CDR-H3 comprises the amino acid sequenceof SEQ ID NO:7.

11. The anti-glyco-MUC1 antibody or antigen-binding fragment of any oneof embodiments 3 to 10, wherein CDR-L1 comprises the amino acid sequenceof SEQ ID NO:30.

12. The anti-glyco-MUC1 antibody or antigen-binding fragment of any oneof embodiments 3 to 10, wherein CDR-L1 comprises the amino acid sequenceof SEQ ID NO:26.

13. The anti-glyco-MUC1 antibody or antigen-binding fragment of any oneof embodiments 3 to 12, wherein CDR-L2 comprises the amino acid sequenceof SEQ ID NO:27.

14. The anti-glyco-MUC1 antibody or antigen-binding fragment of any oneof embodiments 3 to 13, wherein CDR-L3 comprises the amino acid sequenceof SEQ ID NO:10.

15. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 1 or embodiment 2 in which the VH comprises complementaritydetermining regions (CDRs) of SEQ ID NOS:5-7 and the VL comprises CDRsof SEQ ID NOS:8-10.

16. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 1 or embodiment 2 in which the VH comprises complementaritydetermining regions (CDRs) of SEQ ID NOS:23-25 and the VL comprises CDRsof SEQ ID NOS:26, 27, and 10.

17. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 1 or embodiment 2 in which the VH comprises complementaritydetermining regions (CDRs) of SEQ ID NOS:28, 29, and 25 and the VLcomprises CDRs of SEQ ID NOS:30, 9, and 31.

18. The anti-glyco-MUC1 antibody or antigen-binding fragment of any oneof embodiments 1 to 17 which is a chimeric or humanized antibody.

19. The anti-glyco-MUC1 antibody or antigen-binding fragment of any oneof embodiments 1 to 18 in which the VH comprises an amino acid sequencehaving at least 95% sequence identity to SEQ ID NO:3 and the VLcomprises an amino acid sequence having at least 95% sequence identityto SEQ ID NO:4.

20. The anti-glyco-MUC1 antibody or antigen-binding fragment of any oneof embodiments 1 to 18 in which the VH comprises an amino acid sequencehaving at least 97% sequence identity to SEQ ID NO:3 and the VLcomprises an amino acid sequence having at least 97% sequence identityto SEQ ID NO:4.

21. The anti-glyco-MUC1 antibody or antigen-binding fragment of any oneof embodiments 1 to 18 in which the VH comprises an amino acid sequencehaving at least 99% sequence identity to SEQ ID NO:3 and the VLcomprises an amino acid sequence having at least 99% sequence identityto SEQ ID NO:4.

22. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 1 or embodiment 2 in which the VH comprises the amino acidsequence of SEQ ID NO:3 and the VL comprises the amino acid sequence ofSEQ ID NO:4.

23. The anti-glyco-MUC1 antibody or antigen-binding fragment of any ofembodiments 1 to 22 which is multivalent.

24. The anti-glyco-MUC1 antibody or antigen-binding fragment of any ofembodiments 1 to 22 which is in the form of a single-chain variablefragment (scFv).

25. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 24 wherein the scFv comprises the heavy chain variablefragment N-terminal to the light chain variable fragment.

26. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 24 wherein the scFv heavy chain variable fragment and lightchain variable fragment are covalently bound to a linker sequence of4-15 amino acids.

27. The anti-glyco-MUC1 antibody or antigen-binding fragment of any ofembodiments 1 to 22 which is in the form of a multispecific antibody.

28. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 27 wherein the multispecific antibody is a bispecificantibody that binds to a second epitope that is different from the firstepitope.

29. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 28, wherein the bispecific antibody is a CrossMab, a Fab-armexchange antibody, a bispecific T-cell engager (BITE), or adual-affinity retargeting molecule (DART).

30. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 29, wherein the bispecific antibody is a CrossMab.

31. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 30, wherein the bispecific antibody is a CrossMabFAB.

32. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 30, wherein the bispecific antibody is a CrossMab^(VH-VL).

33. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 30, wherein the bispecific antibody is a CrossMab^(CH1-CL).

34. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 29, wherein the bispecific antibody is a Fab-arm exchangeantibody.

35. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 29, wherein the bispecific antibody is a dual-affinityretargeting molecule (DART).

36. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 29, wherein the bispecific antibody is a bispecific T-cellengager (BITE).

37. The anti-glyco-MUC1 antibody or antigen-binding fragment of any oneof embodiments 28 to 35, wherein the second epitope is a MUC1 epitope.

38. The anti-glyco-MUC1 antibody of antigen-binding fragment of any oneof embodiments 28 to 35, wherein the second epitope is a MUC1 epitopethat is overexpressed on cancer cells as compared to normal cells.

39. The anti-glyco-MUC1 antibody or antigen-binding fragment of any oneof embodiments 28 to 36, wherein the second epitope is a T-cell epitope.

40. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 39, wherein the T-cell epitope comprises a CD3 epitope, a CD8epitope, a CD 16 epitope, a CD25 epitope, a CD28 epitope, or an NKG2Depitope.

41. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 40, wherein the T-cell epitope comprises a CD3 epitope, whichis optionally an epitope present in human CD3.

42. The anti-glyco-MUC1 antibody or antigen-binding fragment ofembodiment 41, wherein the CD3 epitope comprises a CD3 gamma epitope, aCD3 delta epitope, a CD3 epsilon epitope, or a CD3 zeta epitope.

43. The anti-glyco-MUC1 antibody or antigen-binding fragment of any oneof embodiments 1 to 42 which is conjugated to a detectable moiety.

44. The anti-glyco-MUC1 antibody or antigen binding fragment ofembodiment 43 in which the detectable marker is an enzyme, aradioisotope, or a fluorescent label.

45. A bispecific antibody comprising a first antigen binding domain thatbinds to CD3 (optionally human CD3) and a second antigen binding domainthat binds to glyco-MUC1, wherein the bispecific antibody competes withan antibody or antigen binding fragment comprising a heavy chainvariable (VH) sequence of SEQ ID NO:3 and a light chain variable (VL)sequence of SEQ ID NO:4 for binding to the breast cancer cell line MCF7or T47D, and wherein the first antigen binding domain comprises a heavychain variable region comprising the heavy chain CDR-H1 of SEQ ID NO:34,the CDR-H2 of SEQ ID NO:35, and the CDR-H3 of SEQ ID NO:36; and a lightchain variable region comprising the light chain CDR-L1 of SEQ ID NO:37,the CDR-L2 of SEQ ID NO:38 and the CDR-L3 of SEQ ID NO:39.

46. The bispecific antibody of embodiment 45, wherein the second antigenbinding domain comprises (i) a heavy chain variable region comprisingthe CDR-H1 of SEQ ID NO: 5, the CDR-H2 of SEQ ID NO: 6, and the CDR-H3of SEQ ID NO: 7; and a light chain variable region comprising the lightchain CDR-L1 of SEQ ID NO: 8, the CDR-L2 of SEQ ID NO: 9 and the CDR-L3of SEQ ID NO:10.

47. The bispecific antibody of embodiment 45 or embodiment 46, whereinthe first antigen binding domain comprises a heavy chain variable regionsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:40 and a light chainvariable region sequence that is at least about 95%, 96%, 97%, 98%, 99%or 100% identical to the amino acid sequence of SEQ ID NO:41.

48. The bispecific antibody of embodiment 47, the first antigen bindingdomain comprises the heavy chain variable region sequence of SEQ IDNO:40 and the light chain variable region sequence of SEQ ID NO:41.

49. The bispecific antibody of any one of embodiments 45 to 48, whereinthe second antigen binding domain comprises a heavy chain variableregion sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:3 and a light chainvariable region sequence that is at least about 95%, 96%, 97%, 98%, 99%or 100% identical to the amino acid sequence of SEQ ID NO:4.

50. The bispecific antibody of embodiment 49, wherein the second antigenbinding domain comprises the heavy chain variable region sequence of SEQID NO:3 and the light chain variable region sequence of SEQ ID NO:4.

51. The bispecific antibody of any one of embodiments 45 to 50, whereinthe first and/or the second antigen binding domain is a Fab molecule.

52. The bispecific antibody of embodiment 51, wherein the first antigenbinding domain is a crossover Fab molecule, wherein either the variableor the constant regions of the Fab light chain and the Fab heavy chainare exchanged.

53. The bispecific antibody of embodiment 52, wherein the first and thesecond antigen binding domain of the bispecific antibody are both Fabmolecules, and in one of the antigen binding domains (particularly thefirst antigen binding domain) the variable domains VL and VH of the Fablight chain and the Fab heavy chain are replaced by each other, wherein

-   -   a. in the constant domain CL of the first antigen binding domain        the amino acid at position 124 is substituted by a positively        charged amino acid (numbering according to Kabat), and wherein        in the constant domain CH1 of the first antigen binding domain        the amino acid at position 147 or the amino acid at position 213        is substituted by a negatively charged amino acid (numbering        according to Kabat EU index); or    -   b. in the constant domain CL of the second antigen binding        domain the amino acid at position 124 is substituted by a        positively charged amino acid (numbering according to Kabat),        and wherein in the constant domain CH1 of the second antigen        binding domain the amino acid at position 147 or the amino acid        at position 213 is substituted by a negatively charged amino        acid (numbering according to Kabat EU index),    -   wherein the constant domains CL and CH1 of the antigen binding        domain having the VH/VL exchange are not replaced by each other.

54. The bispecific antibody of embodiment 53, wherein

-   -   a. in the constant domain CL of the first antigen binding domain        the amino acid at position 124 is substituted independently by        lysine (K), arginine (R) or histidine (H) (numbering according        to Kabat), and in the constant domain CH1 of the first antigen        binding domain the amino acid at position 147 or the amino acid        at position 213 is substituted independently by glutamic acid        (E), or aspartic acid (D) (numbering according to Kabat EU        index); or    -   b. in the constant domain CL of the second antigen binding        domain the amino acid at position 124 is substituted        independently by lysine (K), arginine (R) or histidine (H)        (numbering according to Kabat), and in the constant domain CH1        of the second antigen binding domain the amino acid at position        147 or the amino acid at position 213 is substituted        independently by glutamic acid (E), or aspartic acid (D)        (numbering according to Kabat EU index).

55. The bispecific antibody of embodiment 54, wherein in the constantdomain CL of the second antigen binding domain the amino acid atposition 124 is substituted independently by lysine (K), arginine (R) orhistidine (H) (numbering according to Kabat), and in the constant domainCH1 of the second antigen binding domain the amino acid at position 147or the amino acid at position 213 is substituted independently byglutamic acid (E), or aspartic acid (D) (numbering according to Kabat EUindex).

56. The bispecific antibody of embodiment 55, wherein in the constantdomain CL of the second antigen binding domain the amino acid atposition 124 is substituted independently by lysine (K), arginine (R) orhistidine (H) (numbering according to Kabat), and in the constant domainCH1 of the second antigen binding domain the amino acid at position 147is substituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index).

57. The bispecific antibody of embodiment 55, in the constant domain CLof the second antigen binding domain the amino acid at position 124 issubstituted independently by lysine (K), arginine (R) or histidine (H)(numbering according to Kabat) and the amino acid at position 123 issubstituted independently by lysine (K), arginine (R) or histidine (H)(numbering according to Kabat), and in the constant domain CH1 of thesecond antigen binding domain the amino acid at position 147 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index) and the amino acid at position213 is substituted independently by glutamic acid (E), or aspartic acid(D) (numbering according to Kabat EU index).

58. The bispecific antibody of embodiment 57, wherein in the constantdomain CL of the second antigen binding domain the amino acid atposition 124 is substituted by lysine (K) (numbering according to Kabat)and the amino acid at position 123 is substituted by lysine (K)(numbering according to Kabat), and in the constant domain CH1 of thesecond antigen binding domain the amino acid at position 147 issubstituted by glutamic acid (E) (numbering according to Kabat EU index)and the amino acid at position 213 is substituted by glutamic acid (E)(numbering according to Kabat EU index).

59. The bispecific antibody of embodiment 57, wherein in the constantdomain CL of the second antigen binding domain the amino acid atposition 124 is substituted by lysine (K) (numbering according to Kabat)and the amino acid at position 123 is substituted by arginine (R)(numbering according to Kabat), and in the constant domain CH1 of thesecond antigen binding domain the amino acid at position 147 issubstituted by glutamic acid (E) (numbering according to Kabat EU index)and the amino acid at position 213 is substituted by glutamic acid (E)(numbering according to Kabat EU index).

60. The bispecific antibody of any one of embodiments 53 to 59, whereinthe constant domain CL of the second antigen binding domain is of kappaisotype.

61. The bispecific antibody of any one of embodiments 45 to 60, whereinthe first and the second antigen binding domain are fused to each other,optionally via a peptide linker.

62. The bispecific antibody of embodiment 61, wherein the first and thesecond antigen binding domain are each a Fab molecule and either (i) thesecond antigen binding domain is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the firstantigen binding domain, or (ii) the first antigen binding domain isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the second antigen binding domain.

63. The bispecific antibody of any one of embodiments 45 to 62, whereinthe bispecific antibody provides monovalent binding to CD3.

64. The bispecific antibody of embodiment 63, which comprises twoantigen binding domains that specifically bind to glyco-MUC1.

65. The bispecific antibody of embodiment 64, wherein the two antigenbinding domains that specifically bind to glyco-MUC1 comprise the sameamino acid sequences.

66. The bispecific antibody of any one of embodiments 45 to 65, whereinthe bispecific antibody further comprises an Fc domain composed of afirst and a second subunit.

67. The bispecific antibody of embodiment 66, wherein the Fc domain isan IgG Fc domain.

68. The bispecific antibody of embodiment 67, wherein the Fc domain isan IgG₁ Fc domain.

69. The bispecific antibody of embodiment 67, wherein the Fc domain isan IgG₄ Fc domain.

70. The bispecific antibody of embodiment 69, wherein the IgG4 Fc domaincomprises an amino acid substitution at position S228 (Kabat EU indexnumbering), preferably the amino acid substitution S228P.

71. The bispecific antibody of embodiment 66, wherein the Fc domain is ahuman Fc domain.

72. The bispecific antibody of embodiment 71, wherein the Fc domain is ahuman IgG₁ Fc domain, which optionally comprises SEQ ID NO:42.

73. The bispecific antibody of any one of embodiments 66 to 72, whereinthe first, the second and, where present, the third antigen bindingdomain are each a Fab molecule, and (a) either (i) the second antigenbinding domain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first antigen binding domainand the first antigen binding domain is fused at the C-terminus of theFab heavy chain to the N-terminus of the first subunit of the Fc domain,or (ii) the first antigen binding domain is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding domain and the second antigen binding domain isfused at the C-terminus of the Fab heavy chain to the N-terminus of thefirst subunit of the Fc domain; and (b) the third antigen bindingdomain, where present, is fused at the C-terminus of the Fab heavy chainto the N-terminus of the second subunit of the Fc domain.

74. The bispecific antibody of any one of embodiments 66 to 73, whereinthe Fc domain comprises a modification promoting the association of thefirst and the second subunit of the Fc domain.

75. The bispecific antibody of any one of embodiments 66 to 74, whereinthe Fc domain comprises one or more amino acid substitutions thatreduces binding to an Fc receptor and/or effector function.

76. The bispecific antibody of embodiment 45, comprising

-   -   a. a first antigen binding domain that specifically binds to        CD3, wherein the first antigen binding domain is a crossover Fab        molecule wherein either the variable or the constant regions,        preferably the variable regions, of the Fab light chain and the        Fab heavy chain are exchanged;    -   b. a second and a third antigen binding domain that specifically        bind to glyco-MUC1, comprising a heavy chain variable region        comprising the heavy chain CDR-H1 of SEQ ID NO: 5, the CDR-H2 of        SEQ ID NO: 6, and the CDR-H3 of SEQ ID NO: 7; and a light chain        variable region comprising the light chain CDR-L1 of SEQ ID NO:        8, the CDR-L2 of SEQ ID NO: 9 and the CDR-L3 of SEQ ID NO:10,        wherein the second and third antigen binding domain are each a        Fab molecule;    -   c. an Fc domain composed of a first and a second subunit capable        of stable association,    -   wherein the second antigen binding domain is fused at the        C-terminus of the Fab heavy chain to the N-terminus of the Fab        heavy chain of the first antigen binding domain, and the first        antigen binding domain is fused at the C-terminus of the Fab        heavy chain to the N-terminus of the first subunit of the Fc        domain, and wherein the third antigen binding domain is fused at        the C-terminus of the Fab heavy chain to the N-terminus of the        second subunit of the Fc domain.

77. The bispecific antibody of embodiment 77, wherein the first antigenbinding domain comprises a heavy chain variable region comprising theheavy chain CDR-H1 of SEQ ID NO:34, the CDR-H2 of SEQ ID NO:35, and theCDR-H3 of SEQ ID NO:36; and a light chain variable region comprising thelight chain CDR-L1 of SEQ ID NO:37, the CDR-L2 of SEQ ID NO:38 and theCDR-L3 of SEQ ID NO:39.

78. The bispecific antibody of embodiment 77, wherein the first antigenbinding domain comprises a heavy chain variable region sequence that isat least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO:40 and a light chain variable region sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO:41.

79. The bispecific antibody of embodiment 78, wherein the first antigenbinding domain comprises the heavy chain variable region sequence of SEQID NO:40 and the light chain variable region sequence of SEQ ID NO:41.

80. The bispecific antibody of any one of embodiments 76 to 79, whereinthe second and third antigen binding domain comprise a heavy chainvariable region sequence that is at least about 95%, 96%, 97%, 98%, 99%or 100% identical to the amino acid sequence of SEQ ID NO:3 and a lightchain variable region sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:4.

81. The bispecific antibody of embodiment 80, wherein the second andthird antigen binding domains comprise the heavy chain variable regionof SEQ ID NO:3 and the light chain variable region of SEQ ID NO:4.

82. The bispecific antibody of any one of embodiments 76 to 81, whereinthe Fc domain incorporates, singly or in combination, all of thefeatures described in Sections 6.1 and 6.2 in relation to Fc domains.

83. The bispecific antibody of any one of embodiments 76 to 82, whereinthe antigen binding domains and the Fc region are fused to each other bypeptide linkers.

84. The bispecific antibody of embodiment 83, wherein the peptidelinkers comprise the peptide linkers as in SEQ ID NO:45 and/or SEQ IDNO:46.

85. The bispecific antibody of any one of embodiments 76 to 84, whereinin the constant domain CL of the second and the third Fab molecule, theamino acid at position 124 is substituted by lysine (K) (numberingaccording to Kabat) and the amino acid at position 123 is substituted bylysine (K) or arginine (R), preferably by arginine (R) (numberingaccording to Kabat), and in the constant domain CH1 of the second andthe third Fab molecule under (ii) the amino acid at position 147 issubstituted by glutamic acid (E) (numbering according to Kabat EU index)and the amino acid at position 213 is substituted by glutamic acid (E)(numbering according to Kabat EU index).

86. The bispecific antibody of any one of embodiments 76 to 85, whichcomprises a polypeptide comprising a sequence that is at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ IDNO:43, a polypeptide comprising a sequence that is at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ IDNO:44, a polypeptide comprising a sequence that is at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ IDNO:45, and a polypeptide comprising a sequence that is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ IDNO:46.

87. The bispecific antibody of embodiment 86, wherein the bispecificantibody comprises a polypeptide comprising the sequence of SEQ IDNO:43, a polypeptide comprising the sequence of SEQ ID NO:44, apolypeptide comprising the sequence of SEQ ID NO:45, and a polypeptidecomprising the sequence of SEQ ID NO:46.

88. The bispecific antibody of embodiment 87, which comprises twopolypeptides comprising the sequence of SEQ ID NO:43.

89. The bispecific antibody of any one of embodiments 45 to 88 which isconjugated to a detectable moiety.

90. The bispecific antibody of embodiment 89 in which the detectablemarker is an enzyme, a radioisotope, or a fluorescent label.

91. A fusion protein comprising the amino acid sequence of theanti-glyco-MUC1 antibody or antigen-binding fragment of any ofembodiments 1 to 44 or the bispecific antibody of any one of embodiments45 to 90 operably linked to at least a second amino acid sequence.

92. The fusion protein of embodiment 91, wherein the second amino acidsequence is that of 4-1BB, CD3-zeta, or a fragment thereof.

93. The fusion protein of embodiment 91, wherein the second amino acidsequence is that of a fusion peptide.

94. The fusion protein of embodiment 93, wherein the fusion peptide is aCD28-CD3-zeta or 4-IBB (CD137)-CD3-zeta fusion peptide.

95. The fusion protein of embodiment 91, wherein the second amino acidsequence is that of a modulator of T cell activation or a fragmentthereof.

96. The fusion protein of embodiment 95, wherein the modulator of T cellactivation is IL-15 or IL-15Ra.

97. A chimeric antigen receptor (CAR) comprising the scFv of any one ofembodiments 24 to 26.

98. The CAR of embodiment 97, comprising in amino- to carboxy-terminalorder: a human CD8 leader peptide, the scFv, a human CD8 hinge domain, ahuman CD8 transmembrane domain, and a CD3-zeta signaling domain.

99. An antibody-drug conjugate comprising the anti-glyco-MUC1 antibodyor antigen-binding fragment of any of embodiments 1 to 44 or thebispecific antibody of any one of embodiments 45 to 90 or the fusionprotein of any one of embodiments 91 to 96 conjugated to a cytotoxicagent.

100. The antibody-drug conjugate of embodiment 99, wherein the cytotoxicagent is an auristatin, a DNA minor groove binding agent, an alkylatingagent, an enediyne, a lexitropsin, a duocarmycin, a taxane, adolastatin, a maytansinoid, or a vinca alkaloid.

101. The antibody-drug conjugate of embodiment 100, wherein theanti-glyco-MUC1 antibody or antigen-binding fragment or bispecificantibody is conjugated to the cytotoxic agent via a linker.

102. The antibody-drug conjugate of embodiment 101, wherein the linkeris cleavable under intracellular conditions.

103. The antibody-drug conjugate of embodiment 102, wherein thecleavable linker is cleavable by an intracellular protease.

104. The antibody-drug conjugate of embodiment 103, wherein the linkercomprises a dipeptide.

105. The antibody-drug conjugate of embodiment 104, wherein thedipeptide is val-cit or phe-lys.

106. The antibody-drug conjugate of embodiment 102, wherein thecleavable linker is hydrolyzable at a pH of less than 5.5.

107. The antibody-drug conjugate of embodiment 106, wherein thehydrolyzable linker is a hydrazone linker.

108. The antibody-drug conjugate of embodiment 102, wherein thecleavable linker is a disulfide linker.

109. A nucleic acid comprising a coding region for an anti-glyco-MUC1antibody or antigen-binding fragment of any of embodiments 1 to 44 orthe bispecific antibody of any one of embodiments 45 to 90, the fusionprotein of any one of embodiments 91 to 96, or the CAR of embodiment 97or embodiment 98.

110. The nucleic acid of embodiment 109 in which the coding region iscodon-optimized for expression in a human cell.

111. A vector comprising the nucleic acid of embodiment 109 orembodiment 110. 112. The vector of embodiment 111 which is a viralvector. 113. The vector of embodiment 112 wherein the viral vector is alentiviral vector. 114. A host cell engineered to express the nucleicacid of embodiment 109 or embodiment 110.

115. The host cell of embodiment 114, which is a human T-cell engineeredto express the CAR of embodiment 97 or embodiment 98.

116. A host cell comprising the vector of any one of embodiments 111 to113.

117. The host cell of embodiment 116 which is a T-cell and wherein thevector encodes the CAR of embodiment 97 or embodiment 98.

118. A pharmaceutical composition comprising (a) the anti-glyco-MUC1antibody or antigen binding fragment of any of embodiments 1 to 44, thebispecific antibody of any one of embodiments 45 to 90, the fusionprotein of any one of embodiments 91 to 96, the CAR of embodiment 97 orembodiment 98, the antibody-drug conjugate of any one of embodiments 99to 108, the nucleic acid of embodiment 109 or embodiment 110, the vectorof any one of embodiments 111 to 113, or the host cell of embodiment anyone of embodiments 114 to 117, and (b) a physiologically suitablebuffer, adjuvant or diluent.

119. A method treating cancer comprising administering to a subject inneed thereof an effective amount of the anti-glyco-MUC1 antibody orantigen binding fragment of any of embodiments 1 to 44, the bispecificantibody of any one of embodiments 45 to 90, the fusion protein of anyone of embodiments 91 to 96, the CAR of embodiment 97 or embodiment 98,the antibody-drug conjugate of any one of embodiments 99 to 108, thenucleic acid of embodiment 109 or embodiment 110, the vector of any oneof embodiments 111 to 113, the host cell of embodiment any one ofembodiments 114 to 117, or the pharmaceutical composition of embodiment118.

120. The method of embodiment 119, wherein the subject is suffering frombreast cancer, non-small cell lung cancer, prostate cancer, pancreaticcancer, esophageal cancer, or colorectal cancer.

121. A method of detecting cancer in a biological sample, comprisingcontacting a sample with an anti-glyco-MUC1 antibody or antigen-bindingfragment according to any one of embodiments 1 to 44 and detectingbinding of the anti-glyco-MUC1 antibody or antigen-binding fragment.

122. The method of embodiment 121, further comprising quantitating thebinding of the anti-glyco-MUC1 antibody or antigen-binding fragment.

123. The method of embodiment 121 or embodiment 122, wherein the bindingis compared to a normal tissue control as a negative/baseline controland/or to a cancerous tissue control as a positive control.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.In the event that there is an inconsistency between the teachings of oneor more of the references incorporated herein and the presentdisclosure, the teachings of the present specification are intended.

9. REFERENCES

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2. Fontenot, J. D., Finn, O. J., Dales, N., Andrews, P. C., andMontelaro, R. C. (1993) Synthesis of large multideterminant peptideimmunogens using a poly-proline beta-turn helix motif. Pept. Res., 6,330-336.

3. Mandel, U., Petersen, O. W., Sorensen, H., Vedtofte, P., Hakomori, S.I., Clausen, H., and Dabelsteen, E. (1991) Simple mucin-typecarbohy-drates in oral stratified squamous and salivary-gland epithelia.J. Invest. Dermatol., 97, 713-721.

4. Miles, D. W., Linehan, J., Smith, P., and Filipe, I. (1995)Expression of sialyl-Tn in gastric cancer: correlation with knownprognostic factors. Br. J. Cancer., 71, 1074-1076.

5. Schwientek, T., Bennett, E. P., Flores, C., Thacker, J., Hollmann,M., Reis, C. A., Behrens, J., Mandel, U., Keck, B., Schafer, M. A., andothers. (2002) Functional conservation of subfamilies of putativeUDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferasesin Drosophila, Caenorhabditis elegans, and mammals. One subfamilycomposed of I(2)35Aa is essential in Drosophila. J. Biol. Chem., 277,22623-22638.

6. Soares, R., Marinho, A., and Schmitt, F. (1996) Expression ofSialyl-Tn in breast cancer. Correlation with prognostic parameters.Pathol. Res. Pract., 192,1181-1186.

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8. Werther, J. L., Tatematsu, M., Klein, R., Kurihara, M., Kumagai, K.,Llorens, P., Guidugli, N. J., Bodian, C., Pertsemlidis, D., Yamachika,T., and others. (1996) Sialosyl-Tn antigen as a marker of gastric cancerprogression: an international study. Int. J. Cancer., 69, 193-199.

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1-77. (canceled)
 78. A method of treating cancer comprisingadministering to a subject in need thereof an effective amount of ananti-glyco-MUC1 antibody or antigen binding fragment that: a. comprisesa complementarity determining region (CDR) H1 comprising the amino acidsequence of SEQ ID NO:33, a CDR-H2 comprising the amino acid sequence ofSEQ ID NO:29, a CDR-H3 comprising the amino acid sequence of SEQ IDNO:25, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, aCDR-L2 comprising the amino acid sequence of SEQ ID NO:9, and a CDR-L3comprising the amino acid sequence of SEQ ID NO:31; and b. binds to theMUC1 tandem repeat (VTSAPDTRPAPGSTAPPAHG)₃ (SEQ ID NO:47) that has beenglycosylated in vitro using purified recombinant humanglycosyltransferases GalNAc-T1, GalNAc-T2, and GalNAc-T4 (referred tohereinafter as the “first epitope”).
 79. A method of treating cancercomprising administering to a subject in need thereof an effectiveamount of an anti-glyco-MUC1 antibody or antigen binding fragment that:a. comprises a complementarity determining region (CDR) H1 comprisingthe amino acid sequence of SEQ ID NO:33, a CDR-H2 comprising the aminoacid sequence of SEQ ID NO:29, a CDR-H3 comprising the amino acidsequence of SEQ ID NO:25, a CDR-L1 comprising the amino acid sequence ofSEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ IDNO:9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:31;and b. preferentially binds to a glyco-MUC1 epitope that isoverexpressed on cancer cells as compared to normal cells.
 80. Themethod of claim 78, wherein the anti-glyco-MUC1 antibody orantigen-binding fragment comprises a heavy chain variable (VH) sequencecomprising complementarity determining regions (CDRs) of SEQ ID NOS:5-7and a light chain variable (VL) sequence comprising CDRs of SEQ IDNOS:8-10.
 81. The method of claim 78, wherein the anti-glyco-MUC1antibody or antigen-binding fragment comprises a heavy chain variable(VH) sequence comprising complementarity determining regions (CDRs) ofSEQ ID NOS:23-25 and a light chain variable (VL) sequence comprisingCDRs of SEQ ID NOS:26, 27, and
 10. 82. The method of claim 78, whereinthe anti-glyco-MUC1 antibody or antigen-binding fragment comprises aheavy chain variable (VH) sequence comprising complementaritydetermining regions (CDRs) of SEQ ID NOS:28, 29, and 25 and a lightchain variable (VL) sequence comprising CDRs of SEQ ID NOS:30, 9, and31.
 83. The method of claim 78, wherein the anti-glyco-MUC1 antibody orantigen-binding fragment is a chimeric antibody.
 84. The method of claim78, wherein the anti-glyco-MUC1 antibody or antigen-binding fragment isa humanized antibody.
 85. The method of claim 78, wherein theanti-glyco-MUC1 antibody or antigen-binding fragment comprises a heavychain variable (VH) sequence comprising an amino acid sequence having atleast 95% sequence identity to SEQ ID NO:3 and a light chain variable(VL) sequence comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO:4.
 86. The method of claim 79, whereinthe anti-glyco-MUC1 antibody or antigen-binding fragment comprises aheavy chain variable (VH) sequence comprising an amino acid sequencehaving at least 95% sequence identity to SEQ ID NO:3 and a light chainvariable (VL) sequence comprising an amino acid sequence having at least95% sequence identity to SEQ ID NO:4.
 87. The method of claim 84,wherein the anti-glyco-MUC1 antibody or antigen-binding fragmentcomprises a heavy chain variable (VH) sequence comprising an amino acidsequence having at least 95% sequence identity to SEQ ID NO:3 and alight chain variable (VL) sequence comprising an amino acid sequencehaving at least 95% sequence identity to SEQ ID NO:4.
 88. The method ofclaim 78, wherein the anti-glyco-MUC1 antibody or antigen-bindingfragment comprises a heavy chain variable (VH) sequence comprising theamino acid sequence of SEQ ID NO:3 and a light chain variable (VL)sequence comprising the amino acid sequence of SEQ ID NO:4.
 89. Themethod of claim 78, wherein the anti-glyco-MUC1 antibody orantigen-binding fragment is multivalent.
 90. The method of claim 78,wherein the anti-glyco-MUC1 antibody or antigen-binding fragment is inthe form of a single-chain variable fragment (scFv).
 91. The method ofclaim 78, wherein the anti-glyco-MUC1 antibody or antigen-bindingfragment is in the form of a multispecific antibody.
 92. The method ofclaim 91, wherein the multispecific antibody is a bispecific antibodythat binds to a second epitope that is different from the first epitope.93. The method of claim 92, wherein the bispecific antibody comprises aheavy chain variable (VH) sequence comprising an amino acid sequencehaving at least 95% sequence identity to SEQ ID NO:3 and a light chainvariable (VL) sequence comprising an amino acid sequence having at least95% sequence identity to SEQ ID NO:4.
 94. The method of claim 92,wherein the second epitope is a T-cell epitope.
 95. The method of claim94, wherein the T-cell epitope comprises a CD3 epitope, a CD8 epitope, aCD16 epitope, a CD25 epitope, a CD28 epitope, or an NKG2D epitope. 96.The method of claim 95, wherein the T-cell epitope comprises a CD3epitope.
 97. The method of claim 96, wherein the bispecific antibodycomprises a heavy chain variable (VH) sequence comprising an amino acidsequence having at least 95% sequence identity to SEQ ID NO:3 and alight chain variable (VL) sequence comprising an amino acid sequencehaving at least 95% sequence identity to SEQ ID NO:4.
 98. The method ofclaim 96, wherein the CD3 epitope comprises a CD3 gamma epitope, a CD3delta epitope, a CD3 epsilon epitope, or a CD3 zeta epitope.
 99. Themethod of claim 96, wherein the bispecific antibody comprises an IgG Fcdomain composed of two subunits.
 100. The method of claim 99, whereinthe bispecific antibody comprises a heavy chain variable (VH) sequencecomprising an amino acid sequence having at least 95% sequence identityto SEQ ID NO:3 and a light chain variable (VL) sequence comprising anamino acid sequence having at least 95% sequence identity to SEQ IDNO:4.
 101. The method of claim 96, wherein the bispecific antibodycomprises a CD3 binding domain comprising a heavy chain variable (VH)sequence comprising complementarity determining regions (CDRs) of SEQ IDNOS:34-36 and a light chain variable (VL) sequence comprising CDRs ofSEQ ID NOS:37-39.
 102. The method of claim 101, wherein the bispecificantibody comprises a heavy chain variable (VH) sequence comprising anamino acid sequence having at least 95% sequence identity to SEQ ID NO:3and a light chain variable (VL) sequence comprising an amino acidsequence having at least 95% sequence identity to SEQ ID NO:4.
 103. Themethod of claim 96, wherein the bispecific antibody is a bispecific IgGcomprising a Fab-arm having a domain crossover between heavy and lightchains.
 104. The method of claim 103, wherein the bispecific antibodycomprises a heavy chain variable (VH) sequence comprising an amino acidsequence having at least 95% sequence identity to SEQ ID NO:3 and alight chain variable (VL) sequence comprising an amino acid sequencehaving at least 95% sequence identity to SEQ ID NO:4.
 105. The method ofclaim 96, wherein the MUC1 tandem repeat and CD3 binding portions of thebispecific antibody are in the form of Fabs, wherein the CD3 bindingportion is a crossover Fab whose light and heavy chain variable orconstant regions are exchanged.
 106. The method of claim 105, whereinthe bispecific antibody comprises a heavy chain variable (VH) sequencecomprising an amino acid sequence having at least 95% sequence identityto SEQ ID NO:3 and a light chain variable (VL) sequence comprising anamino acid sequence having at least 95% sequence identity to SEQ IDNO:4.
 107. The method of claim 78, wherein the subject is suffering frombreast cancer, non-small cell lung cancer, prostate cancer, pancreaticcancer, esophageal cancer, or colorectal cancer.
 108. A method detectingcancer in a biological sample, comprising contacting a sample with ananti-glyco-MUC1 antibody or antigen-binding fragment and detectingbinding of the anti-glyco-MUC1 antibody or antigen-binding fragment,wherein the anti-glyco-MUC1 antibody or antigen-binding fragment: a.comprises a complementarity determining region (CDR) H1 comprising theamino acid sequence of SEQ ID NO:33, a CDR-H2 comprising the amino acidsequence of SEQ ID NO:29, a CDR-H3 comprising the amino acid sequence ofSEQ ID NO:25, a CDR-L1 comprising the amino acid sequence of SEQ ID NO:8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:9, and aCDR-L3 comprising the amino acid sequence of SEQ ID NO:31; and b. bindsto the MUC1 tandem repeat (VTSAPDTRPAPGSTAPPAHG)₃ (SEQ ID NO:47) thathas been glycosylated in vitro using purified recombinant humanglycosyltransferases GalNAc-T1, GalNAc-T2, and GalNAc-T4.
 109. A methoddetecting cancer in a biological sample, comprising contacting a samplewith an anti-glyco-MUC1 antibody or antigen-binding fragment anddetecting binding of the anti-glyco-MUC1 antibody or antigen-bindingfragment, wherein the anti-glyco-MUC1 antibody or antigen-bindingfragment: a. comprises a complementarity determining region (CDR) H1comprising the amino acid sequence of SEQ ID NO:33, a CDR-H2 comprisingthe amino acid sequence of SEQ ID NO:29, a CDR-H3 comprising the aminoacid sequence of SEQ ID NO:25, a CDR-L1 comprising the amino acidsequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence ofSEQ ID NO:9, and a CDR-L3 comprising the amino acid sequence of SEQ IDNO:31; and b. preferentially binds to a glyco-MUC1 epitope that isoverexpressed on cancer cells as compared to normal cells.